Method and composition for treating migraines

ABSTRACT

A method for treating migraines is disclosed. The method utilizes a rapid drug delivery system which prevents deactivation or degradation of the active agent, including small molecules and peptides being administered to a patient in need of treatment. In particular, the drug delivery system is designed for inhalation for delivery of drugs to the pulmonary circulation in a rapid and therapeutically effective manner.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/293,017, filed on Nov. 9, 2011, which claims the benefit of U.S.Provisional Application No. 61/412,339, filed Nov. 10, 2010, each ofwhich is incorporated by reference herein in its entirety.

U.S. patent application Ser. No. 13/293,017, filed on Nov. 9, 2011, isalso a continuation-in-part of U.S. patent application Ser. No.12/258,341, filed on Oct. 24, 2008, which claims the benefit of U.S.Provisional Application Nos. 60/982,368 filed Oct. 24, 2007; 60/985,620filed Nov. 5, 2007; 61/022,274 filed Jan. 18, 2008; 61/033,740 filedMar. 4, 2008; 61/052,127 filed May 9, 2008; and 61/094,823 filed Sep. 5,2008. The entire content of each of these applications is incorporatedby reference herein.

U.S. patent application Ser. No. 13/293,017, filed on Nov. 9, 2011, isalso a continuation-in-part of PCT/US11/41303, filed on Jun. 21, 2011,which claims the benefit of U.S. provisional patent application No.61/411,775, filed on Nov. 9, 2010 and U.S. Provisional PatentApplication 61/357,039, filed Jun. 21, 2010. The entire content of eachof these applications is incorporated by reference herein.

TECHNICAL FIELD

Method and composition for treating migraine are disclosed. The methodcomprises administering a pharmaceutical formulation comprising a smallmolecule, including triptans such as sumatriptan to a patient in need oftreatment using a drug delivery system for pulmonary inhalation. Inparticular, the drug delivery system comprises a breath powered drypowder inhaler for oral inhalation.

BACKGROUND

Drug delivery systems for the treatment of disease which introduceactive ingredients into the circulation for the treatment of disease arenumerous and include oral, transdermal, subcutaneous and intravenousadministration. While these systems have been used for quite a long timeand can deliver sufficient medication for the treatment of manydiseases, there are numerous challenges associated with these drugdelivery mechanisms. In particular, delivery of effective amounts ofproteins and peptides to treat a target disease has been problematic.Many factors are involved in introducing the right amount of the activeagent, for example, preparation of the proper drug delivery formulationso that the formulation contains an amount of active agent that canreach its site(s) of action in an effective amount.

The active agent must be stable in the drug delivery formulation and theformulation should allow for absorption of the active agent into thecirculation and remain active so that it can reach the target site(s) ofaction at effective therapeutic levels while minimizing the amount ofdose to be administered. Thus, in the pharmacological arts, drugdelivery systems which can deliver a stable active agent are of utmostimportance.

Making drug delivery formulations therapeutically suitable for treatingdisease depends on the characteristics of the active ingredient or agentto be delivered to the patient. Such characteristics can include in anon-limiting manner solubility, pH, stability, toxicity, release rate,and ease of removal from the body by normal physiologic processes. Forexample, in oral administration, if the agent is sensitive to acid,enteric coatings have been developed using pharmaceutically acceptablematerials which can prevent the active agent from being released in thelow pH (acid) of the stomach. Thus, polymers that are not soluble atacidic pH are used to formulate and deliver a dose containingacid-sensitive agents to the small intestine where the pH is neutral. Atneutral pH, the polymeric coating can dissolve to release the activeagent which is then absorbed into the enteric systemic circulation.Orally administered active agents enter the systemic circulation andpass through the liver. In certain cases, some portion of the dose ismetabolized and/or deactivated in the liver before reaching the targettissues. In some instances, the metabolites can be toxic to the patient,or can yield unwanted side effects.

Similarly, subcutaneous and intravenous administration ofpharmaceutically-active agents is not devoid of degradation andinactivation. With intravenous administration of drugs, the drugs oractive ingredients can also be metabolized, for example in the liver,before reaching the target tissue. With subcutaneous administration ofcertain active agents, including various proteins and peptides, there isadditionally degradation and deactivation by peripheral and vasculartissue enzymes at the site of drug delivery and during travel throughthe venous blood stream. In order to deliver a dose that will yield anacceptable quantity for treating disease with subcutaneous andintravenous administration of an active agent, dosing regimes willalways have to account for the inactivation of the active agent byperipheral and vascular venous tissue and ultimately the liver.

SUMMARY

A method of introducing an active agent into the circulatory system of amammal is disclosed herein. The method comprises a rapid drug deliverysystem which prevents deactivation or degradation of the active agentbeing administered to a patient in need of treatment. In particular, thedrug delivery system is designed for pulmonary drug delivery such as byinhalation, for delivery of active agents to the pulmonary circulationin a therapeutically effective manner. In one embodiment, the drugdelivery system has advantages over other methods of drug delivery, forexample, oral, subcutaneous and intravenous administration of drugproducts such as proteins and peptides that are sensitive to enzymaticdeactivation, or prevents other small molecules from degradation in thelocal peripheral and vascular tissue prior to reaching the target site.

In one embodiment disclosed herein, a method for providing an activeagent to a patient in need thereof is disclosed comprising selecting anactive agent subject to degradation in the patient wherein effectivenessof the active agent is reduced by the degradation; associating theactive agent with a diketopiperazine to produce a pharmaceuticalcomposition suitable for pulmonary inhalation; and providing thepharmaceutical composition to the patient so that the active agentreaches the target site with substantially no degradation ordeactivation in therapeutically effective amounts at lower doses thanstandard dosing with other routes of administration.

Also disclosed herein is a method of treating disease comprisingselecting a patient being treated with or a patient with a conditiontreatable by a labile active agent; providing a composition comprisingthe labile active agent in association with a diketopiperazine; andadministering the composition to the patient via pulmonary inhalation;thereby treating the disease or condition.

In another embodiment, the drug delivery system avoids degradation ofthe active agent from first pass metabolism, wherein the active agent isadministered into the arterial circulation in the lungs and is deliveredto the target organ in therapeutically effective levels, by avoidingdegradation that occurs in venous blood circulation, in peripheraltissue, in the gastrointestinal system or in the liver. In thisembodiment, active agents can be delivered at lower concentrations thanis required through other routes of administration. In a particularembodiment, the active agent is a small molecule including, moleculesthat bind to serotonin receptors and are serotonin receptor agonists. Inone embodiment, the molecules induce vasoconstriction of blood vesselsin the brain, relieve swelling and headaches. In one embodiment, thecompositions are used for the treatment of moderate to severe headachesthat interfere with a subject's performance of daily tasks, and showingsymptoms of nausea, vomiting and sensitivity to light and noise.

In another embodiment, the diketopiperazine is2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl; or apharmaceutically acceptable salt thereof. In another embodiment, thepharmaceutical composition is an inhalable dry powder formulation. Inyet another embodiment, the inhalable dry powder formulation furthercomprises a pharmaceutically acceptable carrier or excipient.

In one embodiment, the inhalable dry powder formulation is provided tothe patient by pulmonary inhalation using a dry powder inhalationsystem. In one embodiment, the system comprises a dry powder inhalerwith or without a container and a dry powder formulation.

In an exemplary embodiment, a method is provided for treating a migraineheadache, which method comprises administering to a patient in need oftreatment a dry powder composition by oral inhalation; wherein the drypowder composition comprises an active agent for treating migraines,including, a triptan such as sumatriptan, almotriptan, eletriptan,frovatriptan, naratriptan, rizatriptan, zolmitriptan andpharmaceutically acceptable salts thereof, and a substituteddiketopiperazine such as fumaryl diketopiperazine, or a salt of thediketopiperazine such as disodium fumaryl diketopiperazine. The drypowder composition can be administered to the patient at the time ofonset of the migraine headache as needed by the patient or as determinedand instructed by the physician. In one embodiment, the dose of thetriptan can reduce or avoid unwanted side effects associated withinjectable or tablet drug therapy, including, flushing, sweating,vertigo, fatigue, tingling, drowsiness, dizziness, dry mouth, heartburn,abdominal pain, abdominal cramps, weakness, feeling of warmth orcoldness, bitter taste from tablets and nasal sprays, and local burningfrom injection site by proving a reduced amount of triptan required withother modes of administration.

In a particular embodiment, a method of treating symptoms associatedwith migraine comprises administering to a subject in need of saidtreatment a thereapeutically effective amount of a dry powderpharmaceutical composition by inhalation comprising a triptan,including, sumatriptan, almotriptan, eletriptan, frovatriptan,naratriptan, rizatriptan, zolmitriptan and pharmaceutically acceptablesalts thereof, and a substituted diketopiperazine in a compositioncomprising bis[3,6-(N-fumaryl-4-aminobutyl)]-2,5-diketopiperazine orbis[3,6-(N-fumaryl-4-aminobutyl)]-2,5-diketopiperazine disodium salt. Incertain embodiments, the pharmaceutical composition can comprise a drypowder comprising a pharmaceutically acceptable carrier or otherinactive agents. In some embodiments, the amount of triptans in the drypowder composition, for example, sumatriptan succinate can varydepending on the subject's requirements, for example, the triptan can bein amounts 1 mg or greater. In example embodiments, the amount ofsumatriptan or a salt thereof, including, sumatriptan succinate in apowder for pulmonary inhalation can be administered in a range of fromabout 1 mg to about 50 mg. In another embodiment, the triptan is a saltof rizatriptan, including, but not limited to benzoate. In otherembodiments, the triptan salts can be, for example, almotriptan malate,frovatriptan succinate, eletriptan hydrobromide, and naratriptanhydrochloride. In certain embodiments, the dry powder composition canoptionally comprise an amino acid such an aliphatic amino acid, forexample, alanine, glycine, leucine, isoleucine, norleucine at amountsranging from about 0.5% to about 30% by weight. In one particularembodiment, the dry powder composition comprises the amino acidL-leucine. In some embodiments, a pharmaceutical composition maycomprise microparticles, wherein a microparticle may comprise 1) adiketopiperazine, and at least one of: a serotonin receptor agonist,such as a triptan, and an aliphatic amino acid. A triptan, and/or analiphatic amino acid may be incorporated into, adhered to, complexedwith, or coated onto a diketopiperazine microparticle. In someembodiments, a diketopiperazine microparticle may be coated with atleast one of a serotonin receptor agonist, such as a triptan, and analiphatic amino acid.

In alternative embodiments, the method of treating a migrane headachecan comprise a combination therapy administering a dry powdercomposition comprising a triptan by oral inhalation, and optionally,administering a second medication or drug, for example, selectiveserotonin reuptake inhibitor such as fluoxetine and duloxetine, whichcan be given by other routes of administration such as oral tablets orinjections. In one embodiment, the combination therapy can comprise adry powder composition comprising the triptan and one or more additionaldrug(s) that can be administered by inhalation.

In another embodiment of the disclosed method, the step of administeringthe composition to the patient comprises pulmonary administration of thedry powder composition by inhalation using a breath powered, dry powderinhaler with or without a container, wherein the container can be acartridge, such as a unit dosing cartridge for a reusable inhaler, or asingle use inhaler. In this and other embodiments, the dry powderinhaler system comprises a high resistance dry powder inhaler having airflow resistance values through its conduits in use of about 0.0065 toabout 0.200 √(κPa)/L per minute, wherein the dry powder inhaler in usehas an air flow distribution of from about 10% to about 30% through thecontainer, which generates peak inhalation pressure differentials ofabout 2 κPa to about 20 κPa, and peak flow rates of between 7 L to about70 L per minute.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean plasma concentration of active glucagon-likepeptide 1 (GLP-1) in subjects treated with an inhalable dry powderformulation containing a GLP-1 dose of 1.5 mg measured at various timesafter inhalation.

FIG. 2A depicts the mean plasma concentration of insulin in subjectstreated with an inhalable dry powder formulation containing a GLP-1 doseof 1.5 mg measured at various times after inhalation.

FIG. 2B depicts the plasma concentration of GLP-1 in subjects treatedwith an inhalable dry powder formulation containing a GLP-1 dose of 1.5mg measured at various times after inhalation compared to subjectstreated with a subcutaneous administration of GLP-1.

FIG. 2C depicts the plasma insulin concentration in subjects treatedwith an inhalable dry powder formulation containing a GLP-1 dose of 1.5mg measured at various times after inhalation compared to subjectstreated with an intravenous GLP-1 dose of 50 μg and subjects treatedwith a subcutaneous GLP-1 dose.

FIG. 3 depicts the mean plasma concentration of the C-peptide insubjects treated with an inhalable dry powder formulation containing aGLP-1 dose of 1.5 mg measured at various times after inhalation.

FIG. 4 depicts the mean plasma concentration of glucose in subjectstreated with an inhalable dry powder formulation containing GLP-1 dosesof 0.05 mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg, measured at varioustimes after inhalation.

FIG. 5 depicts mean plasma insulin concentrations in patients treatedwith an inhalable dry powder formulation containing GLP-1 doses of 0.05mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg. The data shows that insulinsecretion in response to pulmonary GLP-1 administration is dosedependent.

FIG. 6 depicts mean plasma glucagon concentrations in patients treatedwith an inhalable dry powder formulation containing GLP-1 doses of 0.05mg, 0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg.

FIG. 7 depicts the mean plasma exendin concentrations in male ZuckerDiabetic Fatty (ZDF) rats receiving exendin-4/FDKP (fumaryldiketopiperazine) powder by pulmonary insufflation versus subcutaneous(SC) administered exendin-4. The closed squares represent the responsefollowing pulmonary insufflation of exendin-4/FDKP powder. The opensquares represent the response following administration of SC exendin-4.Data are plotted as means±SD.

FIG. 8 depicts changes in blood glucose concentration from baseline inmale ZDF rats receiving either air control, exendin-4/FDKP powder, orGLP-1/FDKP powder by pulmonary insufflation versus subcutaneouslyadministered exendin-4. The graph also shows a combination experiment inwhich the rats were administered by pulmonary insufflation an inhalationpowder comprising GLP-1/FDKP, followed by an inhalation powdercomprising exendin-4/FDKP. In the graph, the closed diamonds representthe response following pulmonary insufflation of exendin-4/FDKP powder.The closed circles represent the response following administration ofsubcutaneous exendin-4. The triangles represent the response followingadministration of GLP-1/FDKP powder. The squares represent the responsefollowing pulmonary insufflation of air alone. The stars represent theresponse given by 2 mg of GLP-1/FDKP given to the rats by insufflationfollowed by a 2 mg exendin-4/FDKP powder administered also byinsufflation.

FIG. 9A depicts the mean plasma oxyntomodulin concentrations in male ZDFrats receiving oxyntomodulin/FDKP powder by pulmonary insufflationversus intravenous (IV) oxyntomodulin. The squares represent theresponse following IV administration of oxyntomodulin alone. The uptriangles represent the response following pulmonary insufflation of 5%oxyntomodulin/FDKP powder (0.15 mg oxyntomodulin). The circles representthe response following pulmonary insufflation of 15% oxyntomodulin/FDKPpowder (0.45 mg oxyntomodulin). The down triangles represent theresponse following pulmonary insufflation of 30% oxyntomodulin/FDKPpowder (0.9 mg oxyntomodulin). Data are plotted as means±SD.

FIG. 9B depicts the cumulative food consumption in male ZDF ratsreceiving 30% oxyntomodulin/FDKP powder (0.9 mg oxyntomodulin) bypulmonary insufflation (1); oxyntomodulin alone (1 mg oxyntomodulin) byIV injection (2); or air control (3).

FIG. 10A depicts the mean plasma oxyntomodulin concentrations in maleZDF rats receiving oxyntomodulin/FDKP powder by pulmonary insufflationversus air control. The squares represent the response followingadministration of air control. The circles represent the responsefollowing pulmonary insufflation of oxyntomodulin/FDKP powder (0.15 mgoxyntomodulin). The up triangles represent the response followingpulmonary insufflation of oxyntomodulin/FDKP powder (0.45 mgoxyntomodulin). The down triangles represent the response followingpulmonary insufflation of oxyntomodulin/FDKP powder (0.9 mgoxyntomodulin). Data are plotted as means±SD.

FIG. 10B depicts data from experiments showing cumulative foodconsumption in male ZDF rats receiving 30% oxyntomodulin/FDKP powder atvarying doses including 0.15 mg oxyntomodulin (1); 0.45 mg oxyntomodulin(2); or 0.9 mg oxyntomodulin (3) by pulmonary insufflation compared toair control (4). Data are plotted as means±SD. An asterisk (*) denotesstatistical significance.

FIG. 11 depicts the glucose values obtained from six fasted Type 2diabetic patients following administration of a single dose of aninhalable dry powder formulation containing GLP-1 at various timepoints.

FIG. 12 depicts the mean glucose values for the group of six fasted Type2 diabetic patients of FIG. 11, in which the glucose values areexpressed as the change of glucose levels from zero time (dosing) forall six patients.

FIG. 13 depicts data obtained from experiments in which ZDF rats wereadministered exendin-4 in a formulation comprising a diketopiperazine ora salt of a diketopiperazine, wherein the exendin-4 was provided byvarious routes of administration (liquid installation (LIS), SC,pulmonary insufflation (INS)) in an intraperitoneal glucose tolerancetest (IPGTT). In one group, rats were treated with exendin-4 incombination with GLP-1 by pulmonary insufflation.

FIG. 14 depicts cumulative food consumption in male ZDF rats receivingair control by pulmonary insufflation, peptide YY(3-36) (PYY) alone byIV injection, PYY alone by pulmonary instillation, 10% PYY/FDKP powder(0.3 mg PYY) by pulmonary insufflation; 20% PYY/FDKP powder (0.6 mg PYY)by pulmonary insufflation. For each group food consumption was measured30 minutes after dosing, 1 hour after dosing, 2 hours after dosing, and4 hours after dosing. Data are plotted mean±SD.

FIG. 15 depicts the blood glucose concentration in female ZDF ratsadministered PYY/FDKP powder by pulmonary insufflation versusintravenously administered PYY at various times following doseadministration.

FIG. 16 depicts mean plasma concentrations of PYY in female ZDF ratsreceiving PYY/FDKP powder by pulmonary insufflation versus intravenouslyadministered PYY. The squares represent the response followingintravenous administration of PYY alone (0.6 mg). The circles representthe response following liquid instillation of PYY alone (1 mg). The downtriangles represent the response following pulmonary insufflation of 20%PYY/FDKP powder (0.6 mg PYY). The up triangles represent the responsefollowing pulmonary insufflation of 10% PYY/FDKP powder (0.3 mg PYY).The left-pointing triangles represent the response following pulmonaryinsufflation of air alone. Data are plotted as ±SD.

FIG. 17 depicts the relative drug exposure and relative bioeffect of thepresent formulations administered by pulmonary inhalation and containinginsulin, exendin, oxyntomodulin or PYY compared to subcutaneous andintravenous administration.

FIG. 18 depicts mean GLP-1 plasma levels in patients administeredvarious inhaled GLP-1 and control formulations.

FIG. 19 depicts plasma insulin levels in patients administered variousinhaled GLP-1 and control formulations.

FIG. 20 depicts gastric emptying in response to an inhaled GLP-1formulation in patients administered various inhaled GLP-1 and controlformulations.

FIG. 21 depicts an HPLC chromatogram of a solution containingsumatriptan-Na₂FDKP spray-dried powders.

FIG. 22 A and B are scanning electron micrographs of three powders madeby the present method comprising sumatriptan-Na₂FDKP. Panels A and Brepresent powder particles without L-leucine. The powder particles with10% leucine (Panel C and D) and the powder particles made with 20%leucine (Panel E and F).

FIG. 23 depicts a graph of dose-normalized sumatriptan concentrations inblood samples following administration of sumatriptan-Na₂FDKP byinsufflation compared to sumatriptan administered by SC injection andsumatriptan nasal spray by instillation for a period of 4 hrs afteradministration.

FIG. 24 depicts a graph showing the mean sumatriptan levels in blood indogs following sumatriptan administered as a nasal spray, sumatriptanadministered intravenously and sumatriptan-Na₂FDKP by insufflationcompared to control dogs exposed to air insufflation.

FIG. 25 depicts a graph of pharmacodynamic data showing the effects ofintravenously administered sumatriptan compared to Imitrex® administeredby nasal instillation and sumatriptan-Na₂FDKP by insufflation on bloodvessel diameter.

DEFINITION OF TERMS

Prior to setting forth the invention, it may be helpful to provide anunderstanding of certain terms that will be used hereinafter:

Active Agents: As used herein “active agent” refers to drugs,pharmaceutical substances and bioactive agents. Active agents can besmall molecules, which are typically less than about 1,000 in molecularweight, do not necessarily have repeated units. Active agents can alsobe organic macromolecules including nucleic acids, synthetic organiccompounds, polypeptides, peptides, proteins, polysaccharides and othersugars, and lipids. Peptides, proteins, and polypeptides are all chainsof amino acids linked by peptide bonds. Peptides are generallyconsidered to be less than 40 amino acid residues, but may include more.Proteins are polymers that typically contain more than 40 amino acidresidues. The term polypeptide as is know in the art and as used herein,can refer to a peptide, a protein, or any other chain of amino acids ofany length containing multiple peptide bonds, though generallycontaining at least 10 amino acids. The active agents can fall under avariety of biological activity classes, such as vasoactive agents,neuroactive agents, hormones, anticoagulants, immunomodulating agents,cytotoxic agents, antibiotics, antiviral agents, antigens, andantibodies. More particularly, active agents may include, in anon-limiting manner, insulin and analogs thereof, growth hormone,parathyroid hormone (PTH), ghrelin, granulocyte macrophage colonystimulating factor (GM-CSF), glucagon-like peptide 1 (GLP-1), andanalogs of such peptides, alkynes, cyclosporins, clopidogrel and PPACK(D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone), antibodies andfragments thereof, including, but not limited to, humanized or chimericantibodies; F(ab), F(ab)₂, or single-chain antibody alone or fused toother polypeptides; therapeutic or diagnostic monoclonal antibodies tocancer antigens, cytokines, infectious agents, inflammatory mediators,hormones, and cell surface antigens. In some instances, the terms “drug”and “active agent” are used interchangeably.

Diketopiperazine: As used herein, “diketopiperazine” or “DKP” includesdiketopiperazines, derivatives, analogs and modifications thereof, inboth the salt and non-salt form of any of the foregoing, falling withinthe scope of the general Formula 1, wherein the ring atoms E₁ and E₂ atpositions 1 and 4 are either O or N and at least one of the side-chainsR₁ and R₂ located at positions 3 and 6 respectively contains acarboxylic acid (carboxylate) group. Compounds according to Formula 1include, without limitation, diketopiperazines, diketomorpholines anddiketodioxanes and their substitution analogs.

Diketopiperazines, in addition to making aerodynamically suitablemicroparticles, can also facilitate the delivery of active agents byrapidly dissolving at physiologic pH thereby releasing the active agentand speeding its absorption into the circulation. Diketopiperazines canbe formed into particles that incorporate a drug or particles onto whicha drug can be adsorbed. The combination of a drug and a diketopiperazinecan impart improved drug stability. These particles can be administeredby various routes of administration. As dry powders these particles canbe delivered by inhalation to specific areas of the respiratory system,depending on particle size. Additionally, the particles can be madesmall enough for incorporation into an intravenous suspension dosageform. Oral delivery is also possible with the particles incorporatedinto a suspension, tablets or capsules.

In one embodiment, the diketopiperazine is3,6-di(fumaryl-4-aminobutyl)-2,5-diketopiperazine (fumaryldiketopiperazine, FDKP). The FDKP can comprise microparticles in itsacid form or salt forms which can be aerosolized or administered in asuspension.

In another embodiment, the DKP is a derivative of3,6-di(4-aminobutyl)-2,5-diketopiperazine, which can be formed by(thermal) condensation of the amino acid lysine. Exemplary derivativesinclude 3,6-di(succinyl-4-aminobutyl)-, 3,6-di(maleyl-4-aminobutyl)-,3,6-di(glutaryl-4-aminobutyl)-, 3,6-di(malonyl-4-aminobutyl)-,3,6-di(oxalyl-4-aminobutyl)-, and3,6-di(fumaryl-4-aminobutyl)-2,5-diketopiperazine. The use of DKPs fordrug delivery is known in the art (see for example U.S. Pat. Nos.5,352,461, 5,503,852, 6,071,497, and 6,331,318, each of which isincorporated herein by reference for all that it teaches regardingdiketopiperazines and diketopiperazine-mediated drug delivery). The useof DKP salts is described in co-pending U.S. Pat. No. 7,820,676, whichis hereby incorporated by reference for all it teaches regardingdiketopiperazine salts. Pulmonary drug delivery using DKP microparticlesis disclosed in U.S. Pat. No. 6,428,771, which is hereby incorporated byreference in its entirety. Further details related to adsorption ofactive agents onto crystalline DKP particles can be found in co-pendingU.S. Pat. Nos. 7,799,344 and 7,803,404, which are hereby incorporated byreference in their entirety.

Drug delivery system: As used herein, “drug delivery system” refers to asystem for delivering one or more active agents.

Dry powder: As used herein, “dry powder” refers to a fine particulatecomposition that is not suspended or dissolved in a propellant, carrier,or other liquid. It is not meant to necessarily imply a complete absenceof all water molecules.

Percent respirable fraction per fill (% RF/Fill): As used herein “%RF/Fill” refers to the amount of powder particles emitted from aninhaler, or drug delivery system, which particles are in the respirablerange and can be smaller than 5.8 μm, normalized by the total amount ofpowder filled into inhaler or drug delivery system. In some embodiments,the inhaler comprises a cartridge for containing the dry powder.

Early phase: As used herein, “early phase” refers to the rapid rise inblood insulin concentration induced in response to a meal. This earlyrise in insulin in response to a meal is sometimes referred to asfirst-phase. In more recent sources first-phase is sometimes used torefer to the more rapid rise in blood insulin concentration of thekinetic profile achievable with a bolus IV injection of glucose indistinction to the meal-related response.

Endocrine disease: The endocrine system is an information signal systemthat releases hormones from the glands to provide specific chemicalmessengers which regulate many and varied functions of an organism,e.g., mood, growth and development, tissue function, and metabolism, aswell as sending messages and acting on them. Diseases of the endocrinesystem include, but are not limited to diabetes mellitus, thyroiddisease, and obesity. Endocrine disease is characterized by dysregulatedhormone release (a productive pituitary adenoma), inappropriate responseto signalling (hypothyroidism), lack or destruction of a gland (diabetesmellitus type 1, diminished erythropoiesis in chronic renal failure),reduced responsiveness to signaling (insulin resistance of diabetesmellitus type 2), or structural enlargement in a critical site such asthe neck (toxic multinodular goiter). Hypofunction of endocrine glandscan occur as result of loss of reserve, hyposecretion, agenesis,atrophy, or active destruction. Hyperfunction can occur as result ofhypersecretion, loss of suppression, hyperplastic, or neoplastic change,or hyperstimulation. The term endocrine disorder encompasses metabolicdisorders.

Excursion: As used herein, “excursion” can refer to blood glucoseconcentrations that fall either above or below a pre-meal baseline orother starting point. Excursions are generally expressed as the areaunder the curve (AUC) of a plot of blood glucose over time. AUC can beexpressed in a variety of ways. In some instances there will be both afall below and rise above baseline creating a positive and negativearea. Some calculations will subtract the negative AUC from thepositive, while others will add their absolute values. The positive andnegative AUCs can also be considered separately. More sophisticatedstatistical evaluations can also be used. In some instances it can alsorefer to blood glucose concentrations that rise or fall outside a normalrange. A normal blood glucose concentration is usually between 70 and110 mg/dL from a fasting individual, less than 120 mg/dL two hours aftereating a meal, and less than 180 mg/dL after eating. While excursion hasbeen described here in terms of blood glucose, in other contexts theterm may be similarly applied to other analytes.

Glucose elimination rate: As used herein, “glucose elimination rate” isthe rate at which glucose disappears from the blood. It is commonlydetermined by the amount of glucose infusion required to maintain stableblood glucose, often around 120 mg/dL during the study period. Thisglucose elimination rate is equal to the glucose infusion rate,abbreviated as GIR.

Hyperglycemia: As used herein, “hyperglycemia” is a higher than normalfasting blood glucose concentration, usually 126 mg/dL or higher. Insome studies hyperglycemic episodes were defined as blood glucoseconcentrations exceeding 280 mg/dL (15.6 mM).

Hypoglycemia: As used herein, “hypoglycemia” is a lower than normalblood glucose concentration, usually less than 63 mg/dL 3.5 mM),Clinically relevant hypoglycemia is defined as blood glucoseconcentration below 63 mg/dL or causing patient symptoms such ashypotonia, flush and weakness that are recognized symptoms ofhypoglycemia and that disappear with appropriate caloric intake. Severehypoglycemia is defined as a hypoglycemic episode that required glucagoninjections, glucose infusions, or help by another party.

In proximity: As used herein, “in proximity,” as used in relation to ameal, refers to a period near in time to the beginning of a meal orsnack.

Microparticles: As used herein, the term “microparticles” includesparticles of generally 0.5 to 100 microns in diameter and particularlythose less than 10 microns in diameter. Various embodiments will entailmore specific size ranges. The microparticles can be assemblages ofcrystalline plates with irregular surfaces and internal voids as istypical of those made by pH controlled precipitation of the DKP acids.In such embodiments the active agents can be entrapped by theprecipitation process or coated onto the crystalline surfaces of themicroparticle. The microparticles can also be spherical shells orcollapsed spherical shells comprising DKP salts with the active agentdispersed throughout. Typically such particles can be obtained by spraydrying a co-solution of the DKP and the active agent. The DKP salt insuch particles can be amorphous. The forgoing descriptions should beunderstood as exemplary. Other forms of microparticles are contemplatedand encompassed by the term.

Obesity: is a condition in which excess body fat has accumulated to suchan extent that health may be negatively affected. Obesity is typicallyassessed by BMI (body mass index) with BMI of greater than 30 kg/m².

Peripheral tissue: As used herein, “peripheral tissue” refers to anyconnective or interstitial tissue that is associated with an organ orvessel.

Periprandial: As used herein, “periprandial” refers to a period of timestarting shortly before and ending shortly after the ingestion of a mealor snack.

Postprandial: As used herein, “postprandial” refers to a period of timeafter ingestion of a meal or snack. As used herein, late postprandialrefers to a period of time 3, 4, or more hours after ingestion of a mealor snack.

Potentiation: Generally, potentiation refers to a condition or actionthat increases the effectiveness or activity of some agent over thelevel that the agent would otherwise attain. Similarly it may referdirectly to the increased effect or activity. As used herein,“potentiation” particularly refers to the ability of elevated bloodinsulin concentrations to boost effectiveness of subsequent insulinlevels to, for example, raise the glucose elimination rate.

Prandial: As used herein, “prandial” refers to a meal or a snack.

Preprandial: As used herein, “preprandial” refers to a period of timebefore ingestion of a meal or snack.

Pulmonary inhalation: As used herein, “pulmonary inhalation” is used torefer to administration of pharmaceutical preparations by inhalation sothat they reach the lungs and in particular embodiments the alveolarregions of the lung. Typically inhalation is through the mouth, but inalternative embodiments in can entail inhalation through the nose.

DETAILED DESCRIPTION

There is disclosed a method for the treatment of a disease or disorderwhich utilizes a drug delivery system that effectively delivers anactive agent to the pulmonary circulation so that the active agententers the pulmonary circulation and can be delivered in a therapeuticamount to the site(s) of action. The methods of treatment of disease ordisorders comprise administering to a patient in need of treatment aformulation which can deliver the active agent directly into thepulmonary circulation, and thereby to the arterial circulation, and canavoid degradation of the active agent such as peptides, by enzymes orother mechanisms in the local peripheral and/or vasculature tissues ofthe lungs. In one embodiment, the method comprises the effectivetherapeutic delivery of active agents using a drug delivery system whichallows for very rapid lung absorption of the active agent into thecirculation and increases its effective bioavailability. In thisembodiment, lower dosages of an active agent can be delivered by thismethod of administration. In similar embodiments effective doses can beachieved where they were not feasible by other modes of administration.

In embodiments herein, there is disclosed a method for the treatment ofdisease, including, endocrine disease, such as diabetes, hyperglycemiaand obesity. The inventors have identified the need to deliver drugsdirectly to the systemic circulation, in particular, the arterialcirculation in a noninvasive fashion so that the drug reaches the targetorgan(s) prior to returning through the venous system. This approach mayparadoxically result in a higher peak target organ exposure to activeagents than would result from a comparable administration via anintravenous, subcutaneous or other parenteral route. A similar advantagecan be obtained versus oral administration as, even with formulationsproviding protection from degradation in the digestive tract, uponabsorption the active agent also enters the venous circulation.

In one embodiment, the drug delivery system can be used with any type ofactive agent that is rapidly metabolized and/or degraded by directcontact with the local degradative enzymes or other degradativemechanisms, for example, oxidation, phosphorylation or any modificationof the molecules including small molecules, proteins or peptides, in theperipheral or vascular venous tissue encountered with other routes ofadministration such as oral, intravenous, transdermal, and subcutaneousadministration. In this embodiment, the method can comprise the step ofidentifying and selecting an active agent which activity is metabolizedor degraded by oral, subcutaneous or intravenous administration. Forexample, due to lability, subcutaneous injection of GLP-1 has not led toeffective levels of GLP-1 in the blood. This contrasts with peptidessuch as insulin which can be delivered effectively by such modes ofadministration. In these embodiments, the method of administering a drugis advantageous for, for example, rapid onset of treatment since thedrug can reach the target organ more rapidly through the arterialcirculation without invasive therapy such as injections.

In certain embodiments, the method of treatment of a disease or disordercomprises the step of selecting a suitable carrier for inhalation anddelivering an active substance to pulmonary alveoli. In this embodiment,the carrier can be associated with one or more active agents to form adrug/carrier complex which can be administered as a composition thatavoids rapid degradation of the active agent in the peripheral andvascular venous tissue of the lung. In one embodiment, the carrier is adiketopiperazine.

The method described herein can be utilized to deliver many types ofactive agents, including small molecules and biologicals. In particularembodiments, the method utilizes a drug delivery system that effectivelydelivers a therapeutic amount of an active agent, including, smallmolecules or peptide hormones, rapidly into the arterial circulation. Inone embodiment, the one or more active agents include, but are notlimited to peptides such as glucagon-like peptide 1 (GLP-1), proteins,lipokines, small molecule pharmaceuticals, nucleic acids and the like,which is/are sensitive to degradation or deactivation; formulating theactive agent into a dry powder composition comprising a diketopiperazineand delivering the active agent(s) into the systemic circulation bypulmonary inhalation using a cartridge and a dry powder inhaler. In oneembodiment, the method comprises selecting a peptide that is sensitiveto enzymes in the local vascular or peripheral tissue of, for example,the dermis, or lungs. The present method allows the active agent toavoid or reduce contact with peripheral tissue, venous or livermetabolism/degradation. In another embodiment, for systemic delivery theactive agent should not have specific receptors in the lungs.

In alternate embodiments, the drug delivery system can also be used todeliver therapeutic peptides or proteins of naturally occurring,recombinant, or synthetic origin for treating disorders or diseases,including, but not limited to adiponectin, cholecystokinin (CCK),secretin, gastrin, glucagon, motilin, somatostatin, brain natriureticpeptide (BNP), atrial natriuretic peptide (ANP), parathyroid hormone,parathyroid hormone related peptide (PTHrP), IGF-1, growth hormonereleasing factor (GHRF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), anti-IL-8 antibodies, IL-8 antagonists includingABX-IL-8; integrin beta-4 precursor (ITB4) receptor antagonist,enkephalins, nociceptin, nocistatin, orphanin FQ2, calcitonin, CGRP,angiotensin, substance P, neurokinin A, pancreatic polypeptide,neuropeptide Y, delta-sleep-inducing peptide, prostaglandins includingPG-12, LTB receptor blockers including, LY29311, BIIL 284, CP105696;vasoactive intestinal peptide; triptans such as sumatriptan andlipokines such as C16:1n7 or palmitoleate or analogs thereof. In yetanother embodiment, the active agent is a small molecule drug,

In one embodiment, the method of treatment is directed to the treatmentof diabetes, hyperglycemia and/or obesity using, for example,formulations comprising glucagon-like peptide 1 (GLP-1), oxyntomodulin(OXN), or peptide YY(3-36) (PYY) either alone or in combination with oneanother, or in combination with one or more other active agents. In oneembodiment, the method of treatment comprises administration of aformulation which does not contain GLP-1.

The incretin hormones GLP-1 and gastric inhibitory polypeptide (GIP) areproduced by the endocrine cells of the intestine following ingestion offood. GLP-1 and GIP stimulate insulin secretion from the beta cells ofthe islets of Langerhans in the pancreas. Only GLP-1 causes insulinsecretion in the diabetic state; however; the GLP-1 protein itself hasbeen ineffective as a clinical treatment for diabetes when delivered byinjection as it has a very short half-life in vivo. Exenatide (BYETTA®),a 39-amino-acid peptide, is an insulin secretagogue with glucoregulatoryeffects which bears a 50% amino acid homology to GLP-1 and has a longerhalf-life in vivo. Exenatide is a synthetic version of exendin-4, ahormone found in the saliva of the Gila monster.

In an exemplary embodiment, a method for treating obesity, diabetesand/or hyperglycemia comprises administering to a patient in need oftreatment a dry powder composition or formulation comprising GLP-1,which stimulates the rapid secretion of endogenous insulin frompancreatic β-cells without causing unwanted side effects such as profusesweating, nausea, and vomiting. In one embodiment, the method oftreating disease can be applied to a patient, including a mammal,suffering with obesity, Type 2 diabetes mellitus and/or hyperglycemia atdosages ranging from about 0.02 to about 3 mg of GLP-1 in theformulation in a single dose. The method of treating hyperglycemia,diabetes, and/or obesity can be designed so that the patient can receiveat least one dose of a GLP-1 formulation in proximity to a meal orsnack. In this embodiment, the dose of GLP-1 can be selected dependingon the patient's requirements. In one embodiment, pulmonaryadministration of GLP-1 can comprise a GLP-1 dose greater than 3 mg forexample, in treating patients with type 2 diabetes.

In embodiments of the invention, the GLP-1 formulation is administeredby inhalation such as by pulmonary administration. In this embodiment,pulmonary administration can be accomplished by providing the GLP-1 in adry powder formulation for inhalation. The dry powder formulation is astable composition and can comprise microparticles which are suitablefor inhalation and which dissolve rapidly in the lung and rapidlydeliver GLP-1 to the pulmonary circulation. Suitable particle sizes forpulmonary administration can be less than 10 μm in diameter, andpreferably less than 5 μm. Exemplary particle sizes that can reach thepulmonary alveoli range from about 0.5 μm to about 5.8 μm in diameter.Such sizes refer particularly to aerodynamic diameter, but often alsocorrespond to actual physical diameter as well. Such particles can reachthe pulmonary capillaries and can avoid extensive contact with theperipheral tissue in the lung. In this embodiment, the drug can bedelivered to the arterial circulation in a rapid manner and avoiddegradation of the active ingredient by enzymes or other mechanismsprior to reaching its target or site of action in the body. In oneembodiment, dry powder compositions for pulmonary inhalation comprisingGLP-1 and FDKP can comprise microparticles wherein from about 35% toabout 75% of the microparticles have an aerodynamic diameter of lessthan 5.8 μm.

The methods of delivery presented in various embodiments of the presentinvention can provide a more direct path to an active agent's site ofaction. Thus in addition to the avoidance of degradation, though in someinstances still in part due to it, the biodistribution of the activeagent can differ from that achieved with modes of delivery that entailabsorption into and travel through the venous circulation prior toreaching sites of action in the body. Thus, sampling of venous blood todetermine active agent concentration may underestimate the concentrationof active agent at a site of action when using embodiments of thepresent disclosure while, in comparison, overestimating it when othermodes of administration are used. The more labile an active agent is thegreater this effect can be. For active agents such as GLP-1, withmultiple effects and sites of action, a different constellation ofeffects may be observed as the relative concentrations at differentsites of action will differ from that achieved using other modes ofadministration. This can further contribute to greater effectivebioavailability, avoidance of unwanted effects and the like.

In one embodiment, the dry powder formulation for use with the methodscomprises particles comprising a GLP-1 molecule and a diketopiperazineor a pharmaceutically acceptable salt thereof. In this and otherembodiments, the dry powder composition of the present inventioncomprises one or more GLP-1 molecules selected from the group consistingof a native GLP-1, a GLP-1 metabolite, a long acting GLP-1, a GLP-1derivative, a GLP-1 mimetic, an exendin, or an analog thereof. GLP-1analogs include, but are not limited to GLP-1 fusion proteins, such asalbumin linked to GLP-1.

In an exemplary embodiment, the method comprises the administration ofthe peptide hormone GLP-1 to a patient for the treatment ofhyperglycemia and/or diabetes, and obesity. The method comprisesadministering to a patient in need of treatment an effective amount ofan inhalable composition or formulation comprising a dry powderformulation comprising GLP-1 which stimulates the rapid secretion ofendogenous insulin from pancreatic β-cells without causing unwanted sideeffects, including, profuse sweating, nausea, and vomiting. In oneembodiment, the method of treating disease can be applied to a patient,including a mammal, suffering with Type 2 diabetes mellitus and/orhyperglycemia at dosages ranging from about 0.01 mg to about 3 mg, orfrom about 0.2 mg to about 2 mg of GLP-1 in the dry powder formulation.In one embodiment, the patient or subject to be treated is a human. TheGLP-1 can be administered immediately before a meal (preprandially), atmealtime (prandially), and/or at about 15, 30 or 45 minutes after a meal(postprandially). In one embodiment, a single dose of GLP-1 can beadministered immediately before a meal and another dose can beadministered after a meal. In a particular embodiment, about 0.5 mg toabout 1.5 mg of GLP-1 can be administered immediately before a meal,followed by 0.5 mg to about 1.5 mg about 30 minutes after a meal. Inthis embodiment, the GLP-1 can be formulated with inhalation particlessuch as a diketopiperazines with or without additional pharmaceuticalcarriers and excipients. In one embodiment, pulmonary administration ofthe GLP-1 formulation can provide plasma concentrations of GLP-1 greaterthan 100 pmol/L without inducing unwanted adverse side effects, such asprofuse sweating, nausea and vomiting.

In another embodiment, a method for treating a patient including a humanwith type 2 diabetes and hyperglycemia is provided, the method comprisesadministering to the patient an inhalable GLP-1 formulation comprisingGLP-1 in a dose of from about 0.5 mg to about 3 mg in FDKPmicroparticles wherein the levels of glucose in the blood of the patientare reduced to fasting plasma glucose concentrations of from 85 to 70mg/dL within about 20 min after dosing without inducing nausea orvomiting in the patient. In one embodiment, pulmonary administration ofGLP-1 at doses greater than 0.5 mg in a formulation comprising FDKPmicroparticles does not substantially inhibit gastric emptying.

In one embodiment, GLP-1 can be administered either alone as the activeingredient in the composition, or with a dipeptidyl peptidase (DPP-IV)inhibitor such as sitagliptin or vildagliptin, or with one or more otheractive agents. DPP-IV is a ubiquitously expressed serine protease thatexhibits postproline or alanine peptidase activity, thereby generatingbiologically inactive peptides by cleavage at the N-terminal regionafter X-proline or X-alanine, wherein X refers to any amino acid.Because both GLP-1 and GIP (glucose-dependent insulinotropic peptide)have an alanine residue at position 2, they are substrates for DPP-IV.DPP-IV inhibitors are orally administered drugs that improve glycemiccontrol by preventing the rapid degradation of incretin hormones,thereby resulting in postprandial increases in levels of biologicallyactive intact GLP-1 and GIP.

In this embodiment, the action of GLP-1 can be further prolonged oraugmented in vivo if required, using DPP-IV inhibitors. The combinationof GLP-1 and DPP-IV inhibitor therapy for the treatment of hyperglycemiaand/or diabetes allows for reduction in the amount of active GLP-1 thatmay be needed to induce an appropriate insulin response from the β-cellsin the patient. In another embodiment, the GLP-1 can be combined, forexample, with other molecules other than a peptide, such as, forexample, metformin. In one embodiment, the DPP-IV inhibitor or othermolecules, including metformin, can be administered by inhalation in adry powder formulation together with the GLP-1 in a co-formulation, orseparately in its own dry powder formulation which can be administeredconcurrently with or prior to GLP-1 administration. In one embodiment,the DPP-IV inhibitor or other molecules, including metformin, can beadministered by other routes of administration, including orally. In oneembodiment, the DPP-IV inhibitor can be administered to the patient indoses ranging from about 1 mg to about 100 mg depending on the patient'sneed. Smaller concentrations of the DPP-IV inhibitor may be used whenco-administered, or co-formulated with GLP-1. In this embodiment, theefficacy of GLP-1 therapy may be improved at reduced dosage ranges whencompared to current dosage forms.

In embodiments described herein, GLP-1 can be administered at mealtime(in proximity in time to a meal or snack). In this embodiment, GLP-1exposure can be limited to the postprandial period so it does not causethe long acting effects of current therapies. In embodiments wherein aDPP-IV inhibitor is co-administered, the DPP-IV inhibitor may be givento the patient prior to GLP-1 administration at mealtime. The amounts ofDPP-IV inhibitor to be administered can range, for example, from about0.10 mg to about 100 mg, depending on the route of administrationselected. In further embodiments one or more doses of the GLP-1 can beadministered after the beginning of the meal instead of or in additionto a dose administered in proximity to the beginning of a meal or snack.For example one or more doses can be administered 15 to 120 minutesafter the beginning of a meal, such as at 30, 45, 60, or 90 minutes.

In one embodiment, the drug delivery system can be utilized in a methodfor treating obesity so as to control or reduce food consumption in ananimal such as a mammal. In this embodiment, patients in need oftreatment or suffering with obesity are administered a therapeuticallyeffective amount of an inhalable composition or formulation comprisingGLP-1, an exendin, oxyntomodulin, peptide YY(3-36), or combinationsthereof, or analogs thereof, with or without additional appetitesuppressants known in the art. In this embodiment, the method istargeted to reduce food consumption, inhibit food intake in the patient,decrease or suppress appetite, and/or control body weight. In anotherembodiment, the composition does not include GLP-1.

In one embodiment, the inhalable formulation comprises a dry powderformulation comprising the above-mentioned active ingredient with adiketopiperazine, including 2,5-diketo-3,6-di(4-X-aminobutyl)piperazine;wherein X is selected from the group consisting of succinyl, glutaryl,maleyl, and fumaryl, or a salt of the diketopiperazine. In thisembodiment, the inhalable formulation can comprise microparticles forinhalation comprising the active ingredient with the aerodynamiccharacteristics as described above. In one embodiment, the amount ofactive ingredient can be determined by one of ordinary skill in the art,however, the present microparticles can be loaded with various amountsof active ingredient as needed by the patient. For example, foroxyntomodulin, the microparticles can comprise from about 1% (w/w) toabout 75% (w/w) of the active ingredient in the formulation. In certainembodiments, the inhalable formulations can comprise from about 10%(w/w) to about 30% (w/w) of the pharmaceutical composition and can alsocomprise a pharmaceutically acceptable carrier, or excipient, such as asurfactant, such as polysorbate 80. In this embodiment, oxyntomodulincan be administered to the patient from once to about four times a dayor as needed by the patient with doses ranging from about 0.05 mg up toabout 5 mg in the formulation. In particular embodiments, the dosage tobe administered to a subject can range from about 0.1 mg to about 3.0 mgof oxyntomodulin. In one embodiment, the inhalable formulation cancomprise from about 50 pmol to about 700 pmol of oxyntomodulin in theformulation.

In embodiments disclosed herein wherein PYY is used as the activeingredient, a dry powder formulation for pulmonary delivery can be madecomprising from about 0.10 mg to about 3.0 mg of PYY per dose. Incertain embodiments, the formulation can comprise a dry powdercomprising PYY in an amount ranging from about 1% to about 75% (w/w) ofthe peptide in the formulation. In particular embodiments, the amount ofPYY in the formulation can be 5%, 10%, 15%, or 20% (w/w) and furthercomprising a diketopiperazine. In one embodiment, the PYY isadministered in a formulation comprising a diketopiperazine, such asFDKP or a salt thereof, including sodium salts. In certain embodiments,PYY can be administered to a subject in dosage forms so that plasmaconcentrations of PYY after administration are from about 4 pmol/L toabout 100 pmol/L or from about 10 pmol/L to about 50 pmol/L. In anotherembodiment, the amount of PYY can be administered, for example, inamounts ranging from about 0.01 mg to about 30 mg, or from about 5 mg toabout 25 mg in the formulation. Other amounts of PYY can be determinedas described, for example, in Savage et al. Gut 1987 February;28(2):166-70; which disclosure is incorporated by reference herein. ThePYY and/or analog, or oxyntomodulin and/or analog formulation can beadministered preprandially, prandially, periprandially, orpostprandially to a subject, or as needed and depending on the patientphysiological condition.

In one embodiment, the formulation comprising the active ingredient canbe administered to the patient in a dry powder formulation by inhalationusing a dry powder inhaler such as the inhaler disclosed, for example,in U.S. Pat. No. 7,305,986 and U.S. patent application Ser. No.10/655,153 (US 2004/0182387), which disclosures are incorporated hereinby reference. Repeat inhalation of dry powder formulation comprising theactive ingredient can also be administered between meals and daily asneeded. In some embodiments, the formulation can be administered once,twice, three or four times a day.

In still yet a further embodiment, the method of treating hyperglycemiaand/or diabetes comprises the administration of an inhalable dry powdercomposition comprising a diketopiperazine having the formula2,5-diketo-3,6-di(4-X-aminobutyl)piperazine, wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl. In thisembodiment, the dry powder composition can comprise a diketopiperazinesalt. In still yet another embodiment of the present invention, there isprovided a dry powder composition, wherein the diketopiperazine is2,5-diketo-3,6-di-(4-fumaryl-aminobutyl)piperazine, with or without apharmaceutically acceptable carrier, or excipient.

In certain embodiments, the method of treatment can comprise a drypowder formulation for inhalation comprising GLP-1, wherein the GLP-1molecule is native GLP-1, or an amidated GLP-1 molecule, wherein theamidated GLP-1 molecule is GLP-1(7-36) amide, or combinations thereof.In one embodiment, the GLP-1 can be an analog such as exenatide.

In another embodiment, GLP-1 can be administered with insulin as acombination therapy and given prandially for the treatment ofhyperglycemia and/or diabetes, for example, Type 2 diabetes mellitus. Inthis embodiment, GLP-1 and insulin can be co-formulated in a dry powderformulation or administered separately to a patient in their ownformulation. In one embodiment, wherein the GLP-1 and insulin areco-administered, both active ingredients can be co-formulated, forexample, the GLP-1 and insulin can be prepared in a dry powderformulation for inhalation using a diketopiperazine particle asdescribed above. Alternatively, the GLP-1 and insulin can be formulatedseparately, wherein each formulation is for inhalation and comprise adiketopiperazine particle. In one embodiment the GLP-1 and the insulinformulations can be admixed together in their individual powder form tothe appropriate dosing prior to administration. In this embodiment, theinsulin can be short-, intermediate-, or long-acting insulin and can beadministered prandially.

In one embodiment for the treatment of Type 2 diabetes usingco-administration of GLP-1 and insulin, an inhalable formulation ofGLP-1 can be administered to a patient prandially, simultaneously, orsequentially to an inhalable formulation of insulin such asinsulin/FDKP. In this embodiment, in a Type 2 diabetic, GLP-1 canstimulate insulin secretion from the patient's pancreas, which can delaydisease progression by preserving β-cell function (such as by promotingβ-cell growth) while prandially-administered insulin can be used asinsulin replacement which mimics the body's normal response to a meal.In certain embodiments of the combination therapy, the insulinformulation can be administered by other routes of administration. Inthis embodiment, the combination therapy can be effective in reducinginsulin requirements in a patient to maintain the euglycemic state. Inone embodiment, the combination therapy can be applied to patientssuffering with obesity and/or Type 2 diabetes who have had diabetes forless than 10 years and are not well controlled on diet and exercise orsecretagogues. In one embodiment, the patient population for receivingGLP-1 and insulin combination therapy can be characterized by havingβ-cell function greater than about 25% of that of a normal healthyindividual and/or, insulin resistance of less than about 8% and/or canhave normal gastric emptying. In one embodiment, the inhalable GLP-1 andinsulin combination therapy can comprise a rapid acting insulin such asinsulin glulisine (APIDRA®), insulin lispro (HUMALOG®) or insulin aspart(NOVOLOG®), or a long acting insulin such as insulin detemir (LEVEMIR®)or insulin glargine (LANTUS®), which can be administered by aninhalation powder also comprising FDKP or by other routes ofadministration.

In another embodiment, a combination therapy for treating Type 2diabetes can comprise administering to a patient in need of treatment aneffective amount of an inhalable insulin formulation comprising aninsulin and a diketopiperazine, wherein the insulin can be a nativeinsulin peptide, a recombinant insulin peptide, and furtheradministering to the patient a long acting insulin analog which can beprovided by inhalation in a formulation comprising a diketopiperazine orby another route of administration such as by subcutaneous injection.The method can further comprise the step of administering to the patientan effective amount of a DPP IV inhibitor. In one embodiment, the methodcan comprise administering to a patient in need of treatment, aformulation comprising a rapid acting or long acting insulin moleculeand a diketopiperazine in combination with formulation comprising a longacting GLP-1, which can be administered separately and/or sequentially.GLP-1 therapy for treating diabetes, in particular Type 2 diabetes, canbe advantageous since administration of GLP-1 alone in a dry powderinhalable formulation or in combination with insulin or non-insulintherapies can reduce the risk of hypoglycemia.

In another embodiment, rapid acting GLP-1 and a diketopiperazineformulation can be administered in combination with a long acting GLP-1,such as exendin, for the treatment of diabetes, which can be bothadministered by pulmonary inhalation. In this embodiment, a diabeticpatient suffering, for example, with Type 2 diabetes, can beadministered prandially an effective amount of an inhalable formulationcomprising GLP-1 so as to stimulate insulin secretion, whilesequentially or sometime after such as from mealtime up to about 45 min,thereafter administering a dose of exendin-4. Administration ofinhalable GLP-1 can prevent disease progression by preserving β-cellfunction while exendin-4 can be administered twice daily atapproximately 10 hours apart, which can provide basal levels of GLP-1that can mimic the normal physiology of the incretin system in apatient. Both rapid acting GLP-1 and a long acting GLP-1 can beadministered in separate, inhalable formulations. Alternatively, thelong acting GLP-1 can be administered by other methods of administrationincluding, for example, transdermally, intravenously or subcutaneously.In one embodiment, prandial administration of a short-acting and longacting GLP-1 combination may result in increased insulin secretion,greater glucagon suppression and a longer delay in gastric emptyingcompared to long acting GLP-1 administered alone. The amount of longacting GLP-1 administered can vary depending on the route ofadministration. For example, for pulmonary delivery, the long actingGLP-1 can be administered in doses from about 0.1 mg to about 1 mg peradministration, immediately before a meal or at mealtime, depending onthe form of GLP-1 administered to the patient.

In one embodiment, the present method can be applied to the treatment ofobesity. A therapeutically effective amount of an inhalable GLP-1formulation can be administered to a patient in need of treatment,wherein an inhalable dry powder, GLP-1 formulation comprises GLP-1 and adiketopiperazine as described above. In this embodiment, the inhalableGLP-1 formulation can be administered alone or in combination with oneor more endocrine hormone and/or anti-obesity active agents for thetreatment of obesity. Exemplary endocrine hormones and/or anti-obesityactive agents include, but are not limited to, peptide YY,oxyntomodulin, amylin, amylin analogs such as pramlintide acetate, andthe like. In one embodiment, the anti-obesity agents can be administeredin a co-formulation in a dry powder inhalable composition alone or incombination with GLP-1 together or in a separate inhalable dry powdercomposition for inhalation. Alternatively, in the combination of GLP-1with one or more anti-obesity agents, or agents that can cause satiety,the GLP-1 formulation can be administered in a dry powder formulationand the anti-obesity agent can be administered by alternate routes ofadministration. In this embodiment, a DPP-IV inhibitor can beadministered to enhance or stabilize GLP-1 delivery into the pulmonaryarterial circulation. In another embodiment, the DPP-IV inhibitor can beprovided in combination with an insulin formulation comprising adiketopiperazine. In this embodiment, the DPP-IV inhibitor can beformulated in a diketopiperazine for inhalation or it can beadministered in other formulation for other routes of administrationsuch as by subcutaneous injection or oral administration.

In an embodiment, a kit for treating diabetes and/or hyperglycemia isprovided which comprises a medicament cartridge for inhalationcomprising a GLP-1 formulation and an inhalation device which isconfigured to adapt or securely engage the cartridge. In thisembodiment, the kit can further comprise a DPP-IV inhibitorco-formulated with GLP-1, or in a separate formulation for inhalation ororal administration as described above. In variations of thisembodiment, the kit does not include the inhalation device which can beprovided separately.

In one embodiment, the present combination therapy using the drugdelivery system can be applied to treat metabolic disorders orsyndromes. In this embodiment, the drug delivery formulation cancomprise a formulation comprising a diketopiperazine and an activeagent, including GLP-1 and/or a long acting GLP-1 alone or incombination with one or more active agents such as a DPP-IV inhibitorand exendin, targeted to treat the metabolic syndrome. In thisembodiment, at least one of the active agents to be provided to thesubject in need of treatment and who may exhibit insulin resistance canbe administered by pulmonary inhalation.

In another embodiment, the pulmonary administration of an inhalable drypowder formulation comprising GLP-1 and a diketopiperazine can be usedas a diagnostic tool to diagnose the level or degree of progression oftype 2 diabetes in a patient afflicted with diabetes in order toidentify the particular treatment regime suitable for the patient to betreated. In this embodiment, a method for diagnosing the level ofdiabetes progression in a patient identified as having diabetes, themethod comprising administering to the patient a predetermined amount ofan inhalable dry powder formulation comprising GLP-1 and adiketopiperazine and measuring the endogenous insulin production orresponse. The administration of the inhalable dry powder formulationcomprising GLP-1 can be repeated with predetermined amounts of GLP-1until the appropriate levels of an insulin response is obtained for thatpatient to determine the required treatment regime required by thepatient. In this embodiment, if a patient insulin response isinadequate, the patient may require alternative therapies. Patients whoare sensitive or insulin-responsive can be treated with a GLP-1formulation comprising a diketopiperazine as a therapy. In this manner,the specific amount of GLP-1 can be administered to a patient in orderto achieve an appropriate insulin response to avoid hypoglycemia. Inthis and other embodiments, GLP-1 can induce a rapid release ofendogenous insulin which mimics the normal physiology of insulinrelease.

In one embodiment, the present drug delivery system can be applied totreat metabolic disorders or syndromes. In this embodiment, the drugdelivery formulation can comprise a formulation comprising adiketopiperazine and an active agent, including GLP-1 and/or a longacting GLP-1 alone or in combination with one or more active agents suchas a DPP-IV inhibitor and exendin, targeted to treat the metabolicsyndrome. In this embodiment, at least one of the active agents to beprovided to the subject in need of treatment and who may exhibit insulinresistance can be administered by pulmonary inhalation. In anotherembodiment, the drug formulation does not include GLP-1.

In another exemplary embodiment, a method for treating migraines using atherapeutically effective pharmaceutical composition comprising a powderfor pulmonary delivery is disclosed, wherein the powder comprisesmicroparticles of a diketopiperazine and an active agent for treatingmigraines. In this embodiment, the pharmaceutical composition comprises,for example, a diketopiperazine, including, FDKP or an FDKP salt, forexample, a divalent salt of FDKP, including disodium FDKP, and a smallmolecule, including a vasoconstrictor as the active agent Examples ofvasoconstrictors are serotonin receptor agonists including, tripans suchas sumatriptan, almotriptan, eletriptan, frovatriptan, naratriptan,rizatriptan, zolmitriptan and pharmaceutically acceptable salts thereof,including sumatriptan succinate, rizatriptan benzoate, almotriptanmalate. In one embodiment, the vasoconstrictor, for example, a triptancan be provided to a patient in need of treatment in amounts rangingfrom at least about 0.1 mg, at least about 1 mg, at least about 5 mg,about 50 mg or less, about 40 mg or less, about 1 mg to about 50 mg,about 5 mg to about 30 mg, about 10 mg. to about 20 mg, about 1 mg,about 10 mg, about 20 mg, or any amount in a range bounded by, orbetween, any of these values. A pharmaceutical composition comprising atriptan may be given regularly, including daily, twice daily, thricedaily, etc., and/or may be given as need at the onset of migrainesymptoms. In one embodiment, the triptan can be administered to thepatient by inhalation. In a particular embodiment, the triptan isprovided to a patient by oral inhalation for delivery to the arterialcirculation in the lungs.

In an exemplary embodiment of the invention, the drug deliveryformulation can comprise an aliphatic amino acid, for example, alanine,glycine, leucine, isoleucine, norleucine, and serine. In certainembodiments, the aliphatic amino acid is from about 0.5% to about 30% byweight of the composition. In a particular embodiment, thepharmaceutical composition comprises L-leucine. In one embodiment, thepharmaceutical composition comprises a dry powder for oral inhalationcomprising FDKP disodium salt, sumatriptan and L-leucine.

EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples elucidate representativetechniques that function well in the practice of the present invention.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention.

Example 1 Administration of GLP-1 in an Inhalable Dry Powder to HealthyAdult Males

GLP-1 has been shown to control elevated blood glucose in humans whengiven by intravenous (iv) or subcutaneous (sc) infusions or by multiplesubcutaneous injections. Due to the extremely short half-life of thehormone, continuous subcutaneous infusion or multiple daily subcutaneousinjections would be required to achieve clinical efficacy. Neither ofthese routes is practical for prolonged clinical use. Applicants havefound in animal experiments that when GLP-1 was administered byinhalation, therapeutic levels could be achieved. The results of thesestudies can be found, for example, in U.S. patent application Ser. No.11/735,957, the disclosure of which is incorporated by reference herein.

In healthy individuals, several of the actions of GLP-1, includingreduction in gastric emptying, increased satiety, and suppression ofinappropriate glucagon secretion appear to be linked to the burst ofGLP-1 released as meals begin. By supplementing this early surge inGLP-1 with a formulation of GLP-1 and2,5-diketo-3,6-di(4-fumaryl-aminobutyl)piperazine (FDKP) as aninhalation powder, a pharmacodynamic response, including endogenousinsulin production, reduction in glucagon and glucose levels, indiabetic animals can be elicited. In addition, the late surge in nativeGLP-1 linked to increased insulin secretion can be mimicked bypostprandial administration of GLP-1/FDKP inhalation powder.

A Phase 1a clinical trial of GLP-1/FDKP inhalation powder was designedto test the safety and tolerability of selected doses of a new inhaledglycemic control therapeutic product for the first time in humansubjects. GLP-1/FDKP inhalation powder was administered using theMedTone® Inhaler device, previously tested. The experiments weredesigned to identify the safety and tolerability of various doses ofGLP-1/FDKP inhalation powder by pulmonary inhalation. Doses wereselected for human use based on animal safety study results fromnon-clinical studies in rats and primates using GLP-1/FDKP administeredby inhalation as described in U.S. application Ser. No. 11/735,957,which is incorporated herein by reference.

Twenty-six subjects were enrolled into 5 cohorts to provide up to 4evaluable subjects in each of cohorts 1 and 2 and up to 6 evaluablesubjects in each of cohorts 3 to 5 who met eligibility criteria andcompleted the study. Each subject was dosed once with GLP-1 asGLP-1/FDKP inhalation powder at the following dose levels: cohort 1:0.05 mg; cohort 2: 0.45 mg; cohort 3: 0.75 mg; cohort 4: 1.05 mg andcohort 5: 1.5 mg of GLP-1. Dropouts were not replaced. These dosagesassumed a body mass of 70 kg. Persons of ordinary skill in the art candetermine additional dosage levels based on the studies disclosedherein.

In these experiments, the safety and tolerability of ascending doses ofGLP-1/FDKP inhalation powder in healthy adult male subjects weredetermined. The tolerability of ascending doses of GLP-1/FDKP inhalationpowder were determined by monitoring pharmacological or adverse effectson variables including reported adverse events (AE), vital signs,physical examinations, clinical laboratory tests and electrocardiograms(ECG).

Additional pulmonary safety and pharmacokinetic parameters were alsoevaluated. Pulmonary safety as expressed by the incidence of pulmonaryand other adverse events and changes in pulmonary function between Visit1 (Screening) and Visit 3 (Follow-up) was studied. Pharmacokinetic (PK)parameters of plasma GLP-1 and serum fumaryl diketopiperazine (FDKP)following dosing with GLP-1/FDKP inhalation powder were measured asAUC_(0-120 min) plasma GLP-1 and AUC_(0-480 min) serum FDKP. AdditionalPK parameters of plasma GLP-1 included the time to reach maximal plasmaGLP-1 concentration, T_(max) plasma GLP-1; the maximal concentration ofGLP-1 in plasma, C_(max) plasma GLP-1, and the half of total time toreach maximal concentration of GLP-1 in plasma, T_(1/2) plasma GLP-1.Additional PK parameters of serum FDKP included T_(max) serum FDKP,C_(max) serum FDKP, and T_(1/2) serum FDKP. Clinical trial endpointswere based on a comparison of the following pharmacological and safetyparameters determined in the trial subject population. Primary endpointsincluded the incidence and severity of reported AEs, including cough anddyspnea, nausea and/or vomiting, as well as changes from screening invital signs, clinical laboratory tests and physical examinations.Secondary endpoints included pharmacokinetic disposition of plasma GLP-1and serum FDKP (AUC_(0-120 min) plasma GLP-1 and AUC_(0-480 min) serumFDKP), plasma GLP-1 (T_(max) plasma GLP-1, C_(max) plasma GLP-1 T_(1/2)plasma GLP-1); serum FDKP (T_(max) serum FDKP, C_(max) serum FDKP);pulmonary function tests (PFTs), and ECG.

The clinical trial consisted of 3 clinic visits: 1) One screening visit(Visit 1); 2) One treatment visit (Visit 2); and 3) One follow-up visit(Visit 3) 8-14 days after Visit 2. A single dose of GLP-1/FDKPinhalation powder was administered at Visit 2.

Five doses of GLP-1/FDKP inhalation powder (0.05, 0.45, 0.75, 1.05 and1.5 mg of GLP-1) were assessed. To accommodate all doses, formulatedGLP-1/FDKP was mixed with FDKP inhalation powder containing particleswithout active agent. Single-dose cartridges containing 10 mg dry powderconsisting of GLP-1/FDKP inhalation powder (15% weight to weightGLP-1/FDKP) as is or mixed with the appropriate amount of FDKPinhalation powder was used to obtain the desired dose of GLP-1 (0.05 mg,0.45 mg, 0.75 mg, 1.05 mg and 1.5 mg). The first 2 lowest dose levelswere evaluated in 2 cohorts of 4 subjects each and the 3 higher doselevels were evaluated in 3 cohorts of 6 subjects each. Each subjectreceived only 1 dose at 1 of the 5 dose levels assessed. In addition toblood drawn for GLP-1 (active and total) and FDKP measurements, sampleswere drawn for glucagon, glucose, insulin, and C-peptide determination.The results from these experiments are described with reference to thefollowing figures and tables.

FIG. 1 depicts the active GLP-1 plasma concentration in cohort 5 afterpulmonary administration of 1.5 mg of GLP-1 dose. The data showed thatthe peak GLP-1 concentration occurred prior to the first sampling pointat 3 minutes, closely resembling intravenous (IV) bolus administration.GLP-1 plasma concentrations in some subjects were greater than 500pmol/L, the assay limit. Peak active GLP-1 plasma concentrations rangefrom about 150 pmol/L to about 500 pmol/L. Intravenous bolusadministration of GLP-1 as reported in the literature (Vilsboll et al.2000) results in ratios of total:active GLP-1 of 3.0-5.0 compared to aratio of 1.5 in cohort 5 of this study. At comparable activeconcentrations the metabolite peaks were 8-9 fold greater followingintravenous administration compared to pulmonary administration,suggesting that pulmonary delivery results in rapid delivery and lessdegradation of GLP-1.

TABLE 1 Treatment 0.05 mg 0.45 mg 0.75 mg 1.05 mg 1.5 mg Parameter^(a)(n = 4) (n = 4) (n = 6) (n = 6) (n = 6) GLP-1^(a) AUC₀₋₁₂₀ ND n = 1 n =6 n = 4 n = 4 (min*pmol/L) 355.33 880.12 1377.88 AULQ (195.656)(634.054) C_(max) n = 4 n = 4 n = 6 n = 6 n = 6 (pmol/L) 2.828 24.63081.172 147.613 310.700 (2.4507) (8.7291) (63.3601) (122.7014 (54.2431)t_(max) n = 4 n = 4 n = 6 n = 6 n = 6 (min) 3.00 3.00 3.00 3.00 3.00(3.00, 3.00) (3.00, 4.02) (3.00, 6.00) (3.00, 4.98) (3.00, 3.00) T_(1/2)n = 1 n = 3 n = 6 n = 4 n = 6 (min) 6.1507 3.0018 5.5000 3.6489 3.9410(0.83511) (2.96928) (1.88281) (1.79028) FDKP AUC₀₋₁₂₀ n = 6 n = 6(min*pmol/L) 22169.2 25594.7 (4766.858) (5923.689) C_(max) n = 6 n = 6(pmol/L) 184.21 210.36 (56.893) (53.832) t_(max) n = 6 n = 6 (min) 4.506.00 (3.00, 25.02) (3.00, 19.98) T_(1/2) n = 6 n = 6 (min) 126.71 123.82(11.578) (15.640) ^(a)All parameters are mean (SD) except tmax, which ismedian (range) AULQ - Two or more subjects in the dose group had plasmaconcentrations of the analyte that were AULQ; NA = The pharmacokineticprofile did not meet the specifications for this profile because of theshort sampling time (20 minutes); ND = Parameter could not be calculatedbecause of insufficient data is some subjects.

In healthy individuals, physiological post-prandial venous plasmaconcentrations of GLP-1 typically range from 10-20 pmol/L (Vilsboll etal. J. Clin. Endocr. & Metabolism. 88(6):2706-13, June 2003). Theselevels were achieved with some subjects in cohort 2, who received 0.45mg GLP-1. Higher doses of GLP-1 produced peak plasma GLP-1concentrations substantially higher than physiological peak venousconcentrations. However, because the half-life of GLP-1 is short (about1-2 min), plasma concentrations of active GLP-1 fell to thephysiological range by 9 min after administration. Although the peakconcentrations are much higher than those seen physiologically in thevenous circulation, there is evidence that local concentrations of GLP-1may be much higher than those seen systemically.

Table 1 shows the pharmacokinetic profile of GLP-1 in a formulationcomprising FDKP from this study.

FDKP pharmacokinetic parameters are also represented in Table 1 forcohorts 4 and 5. Other cohorts were not analyzed. The data also showsthat mean plasma concentration of FDKP for the 1.05 mg and the 1.5 mgGLP-1 treated subjects were about 184 and 211 pmol/L, respectively.Maximal plasma FDKP concentrations were attained at about 4.5 and 6 minafter administration for the respective dose with a half-life about 2 hr(127 and 123 min).

FIG. 2A depicts mean insulin concentrations in subjects treated with aninhalable dry powder formulation of GLP-1 at a dose of 1.5 mg. The datashow the 1.5 mg GLP-1 dose induced endogenous insulin release fromβ-cells since insulin concentrations were detected in all subjects, andthe mean peak insulin concentrations of about 380 pmol/L occurred at 6min after dosing or earlier. The insulin release was rapid, but notsustained, since plasma insulin concentration fell rapidly after theinitial response to GLP-1. FIG. 2B depicts the GLP-1 plasmaconcentration of subjects treated with the 1.5 mg dose of GLPadministered by pulmonary inhalation compared to subcutaneousadministration of a GLP-1 dose. The data illustrate that pulmonaryadministration of GLP-1 occurs relatively fast and peak plasmaconcentration of GLP-1 occur faster than with subcutaneousadministration. Additionally, pulmonary inhalation of GLP-1 leads toGLP-1 plasma concentrations returning to basal levels much faster thanwith subcutaneous administration. Thus the exposure of the patient toGLP-1 provided by pulmonary inhalation using the present drug deliverysystem is shorter in time than by subcutaneous administration and thetotal exposure to GLP-1 as measured by AUC is less for the inhaledGLP-1. FIG. 2C illustrates that pulmonary administration of a dry powderformulation of GLP-1 induces an insulin response which is similar to theresponse obtained after intravenous administration of GLP-1, butdifferent in peak time and amount of endogenous insulin produced thanwith subcutaneous GLP-1 administration, which indicates that pulmonaryadministration of GLP-1 using the present formulation is moreefficacious at inducing an insulin response.

FIG. 3 depicts the plasma C-peptide concentrations in subjects treatedwith an inhalable dry powder formulation containing a GLP-1 dose of 1.5mg measured at various times after inhalation. The data demonstrate thatC-peptide is released following GLP-1 inhalation confirming endogenousinsulin release.

FIG. 4 depicts fasting plasma glucose concentrations in subjects treatedwith the GLP-1 formulation containing GLP-1. Mean fasting plasma glucose(FPG) concentrations were approximately 4.7 mmol/L for those receivingthe 1.5 mg dose. GLP-1 mediated insulin release is glucose dependent.Hypoglycemia is not historically observed in euglycemic subjects. Inthis experiment, the data clearly show that glucose concentrations inthese euglycemic healthy subjects were reduced following pulmonaryadministration of GLP-1. At the 1.5 mg GLP-1 dose, two of the sixsubjects had glucose concentrations lowered by GLP-1 to below 3.5mmol/L, the laboratory value that defines hypoglycemia. Plasma glucosedecreased more than 1.5 mol/L in two of the six subjects who receivedthe 1.5 mg GLP-1 dose. Moreover, decreases in plasma glucose werecorrelated to the GLP-1 dose. The smallest decrease in glucoseconcentration was seen with the 0.05 mg dose, and the largest decreasewas seen with the 1.5 mg dose. The three intermediate doses of GLP-1produced roughly equal decreases in plasma glucose. The data indicatethat the GLP-1 glucose-dependency was overcome based on GLP-1concentrations above the physiologic range. Physiologic ranges for GLP-1(7-36) amide in normal individuals has been reported to be in the rangeof 5-10 pmol/L during fasting, and increase rapidly after eating to 15to 50 pmol/L (Drucker, D. and Nauck, M. The Lancet 368:1696-1705, 2006).

FIG. 5 further depicts the dose dependent insulin concentrations inplasma after pulmonary administration of GLP-1. In most subjects, theinsulin release was not sustained, since plasma insulin concentrationfell rapidly after the initial response to GLP-1 administration. In mostsubjects, the peak plasma insulin response ranged from 200-400 pmol/Lwith one subject exhibiting peak plasma insulin levels that exceeded 700pmol/L. Thus, the data indicate that insulin response is GLP-1 dosedependent.

FIG. 6 depicts glucagon concentrations in plasma after GLP-1 pulmonaryadministration at the various dosing groups. Baseline glucagon levelsranged from 13.2 pmol/L to 18.2 pmol/L in the various dose groups. Themaximum change in plasma glucagon was seen at 12 min after dosing. Thelargest decrease in plasma glucagon was approximately 2.5 pmol/L and wasseen in the 1.5 mg dose group. The maximum suppression of glucagonsecretion was potentially underestimated because the minima did notalways occur at 12 min.

Tables 2 and 3 report the adverse events or side effect symptomsrecorded for the patient population in the study. The list of adverseevents reported in the literature for GLP-1 administered by injection isnot extensive; and those reported have been described as mild ormoderate, and tolerable. The primary adverse events reported have beenprofuse sweating, nausea and vomiting when active GLP-1 concentrationsexceed 100 pmol/L. As shown in Tables 1 and 3, and FIG. 1, pulmonaryadministration at doses of 1.05 mg and 1.5 mg resulted in active GLP-1concentrations greatly exceeding 100 pmol/L without the side effectsnormally observed with parenteral (subcutaneous, intravenous [eitherbolus or infusion]) GLP-1. None of the subjects in this study reportedsymptoms of nausea, profuse sweating or vomiting. Subjects in Cohort 5reached C_(max) comparable to that observed with a 50 μg/kg IV bolusdata (reported by Vilsboll et al. 2000), where the majority of subjectsreported significant adverse events.

TABLE 2 Adverse Events 0.05 mg 0.45 mg 0.75 mg 1.05 mg 1.5 mg AdverseEvent (n = 4) (n = 4) (n = 6) (n = 6) (n = 6) Cough 3 1 3 5 5 Dysphonia2 — 2 3 3 Productive Cough — — 1 — — Throat Irritation — — — 1 —Headache 1 1 — 1 1 Dizziness — — — — 2 Dysgeusia — — 1 — — Fatigue — — 11 1 Seasonal Allergy — — — 1 — Rhinitis — — — 1 — Increased Appetite — —— — 1

TABLE 3 Comparative Adverse Events of GLP-1: IV vs. PulmonaryAdministration IV^(†) IV^(†)* Pulmonary* Adverse Events (16.7 μg) (50μg) (1.5 mg) Reduced well-being 42% 100%  17%  Nausea 33% 83% 0% Profusesweating 17% 67% 0% ^(†)Vilsboll et al. Diabetes Care, June 2000;*Comparable C_(max)

Tables 2 and 3 show there were no serious or severe adverse eventsreported by any subjects in the study who received GLP-1 by pulmonaryinhalation. The most commonly reported adverse events were thoseassociated with inhalation of a dry powder, cough and throat irritation.Surprisingly, in the patients treated by pulmonary inhalation, nosubject reported nausea or dysphoria, and there was no vomitingassociated with any of these subjects. The inventors also found thatpulmonary administration of GLP-1 in a dry powder formulation lackinhibition of gastric emptying in the above subjects (data not shown).Inhibition of gastric emptying is a commonly encountered unwanted sideeffect associated with injected standard formulations of GLP-1.

In summary, the clinical GLP-1/FDKP powder contained up to 15 wt % GLP-1providing a maximum dose of 1.5 mg GLP-1 in 10 mg of powder. Andersencascade measurements indicated that 35-70% of the particles hadaerodynamic diameters <5.8 μm. A dose of 1.5 mg GLP-1 produced mean peakconcentrations >300 pmol/L of active GLP-1 at the first sampling time (3min); resulted in mean peak insulin concentrations of 375 pmol/L at thefirst measured time point (6 min); reduced mean fasting plasma glucosefrom 85 to 70 mg/dL 20 min after dosing; and was well tolerated and didnot cause nausea or vomiting.

Example 2 Comparison of Pulmonary Administration of GLP-1 and Exenatide,and Subcutaneous Administration of Exenatide to Male Zucker Diabetic FatRats

Much effort has been expended in developing analogs of GLP-1 with longercirculating half-lives to arrive at a clinically useful treatment. Asdemonstrated herein, pulmonary administration of GLP-1 also providesclinically meaningful activity. It was thus of interest to compare thesetwo approaches.

Preparation of FDKP Particles.

Fumaryl diketopiperazine (FDKP) and polysorbate 80 were dissolved indilute aqueous ammonia to obtain a solution containing 2.5 wt % FDKP and0.05 wt % polysorbate 80. The FDKP solution was then mixed with anacetic acid solution containing polysorbate 80 to form particles. Theparticles were washed and concentrated by tangential flow filtration toachieve approximately 11% solids by weight.

Preparation of GLP-1 Stock Solution.

A 10 wt % GLP-1 stock solution was prepared in deionized water bycombining 60 mg GLP-1 solids (86.6% peptide) with 451 mg deionizedwater. About 8 μL glacial acetic acid was added to dissolve the peptide.

Preparation of GLP-1/FDKP Particles.

A 1 g portion of the stock FDKP suspension (108 mg particles) wastransferred to a 2 mL polypropylene tube. The appropriate amount ofGLP-1 stock solution was added to the suspension and gently mixed. ThepH of the suspension was adjusted from pH ˜3.5 to pH ˜4.5 by adding 1 μLaliquots of 50% (v/v) ammonium hydroxide. The GLP-1/FDKP particlesuspension was then pelleted into liquid nitrogen and lyophilized. Thedry powders were assayed by high performance liquid chromatography(HPLC) to confirm drug content.

Preparation of Exenatide Stock Solution.

A 10 wt % exendin stock solution was prepared in 2% wt acetic acid bycombining 281 mg exendin solids (88.9% peptide) with 2219 mg 2% wtacetic acid.

Preparation of Exenatide/FDKP Particles.

A 1533 mg portion of a stock FDKP particle suspension (171 mg particles)was transferred to a 4 mL glass vial. A 304 mg portion of exendin stocksolution was added to the suspension and gently mixed. The pH of thesuspension was adjusted from pH ˜3.7 to pH ˜4.5 by adding 3-5 μLaliquots of 25% (v/v) ammonium hydroxide. The exenatide/FDKP particlesuspension was then pelleted into liquid nitrogen and lyophilized. Thedry powders were assayed by high performance liquid chromatography(HPLC) to confirm drug content.

Pharmacokinetic and Pharmacodynamic Assessment in Rats.

Male Zucker Diabetic Fatty (ZDF) rats (5/group) were assigned to one offour test groups. Animals were fasted overnight then administeredglucose (1 g/kg) by intraperitoneal injection immediately prior to testarticle dosing. Animals in the control group received air by pulmonaryinsufflation. Animals in Group 1 received exenatide (0.3 mg) in saline(0.1 mL) by subcutaneous (SC) injections. Animals in Group 2 received15% by weight exenatide/FDKP (2 mg) by pulmonary insufflation. Animalsin Group 3 received 15% by weight GLP-1/FDKP (2 mg) by pulmonaryinsufflation. Blood samples were collected from the tail prior to dosingand at 15, 30, 45, 60, 90, 120, 240, and 480 min after dosing. Plasmawas harvested. Blood glucose and plasma GLP-1 or plasma exenatideconcentrations were determined.

Exenatide pharmacokinetics are reported in FIG. 7A. These data showedthat exanetide is absorbed rapidly following insufflation ofexenatide/FDKP powder. The bioavailability of the inhaled exenatide was94% compared to subcutaneous injection. This may indicate that pulmonaryadministration is particularly advantageous to exenatide. The time tomaximum peak circulating exenatide concentrations (T_(max)) was 30 minin rats receiving subcutaneous exenatide compared to <15 min in ratsreceiving inhaled exenatide. This T_(max) was similar to that ofinsufflated GLP-1/FDKP (data not shown).

Comparative pharmacodynamics are reported in FIG. 8. These data showedthe changes in blood glucose for all four test groups. Glucoseexcursions following the glucose tolerance test were lower in animalsreceiving inhaled exenatide/FDKP as compared to animals receiving SCexenatide. Since exenatide exposure was comparable in both groups (FIG.7), these data suggest that the shorter time to peak exenatideconcentrations in the exenatide/FDKP group provided better glucosecontrol. Additionally, glucose excursions were comparable in animalsreceiving either GLP-1/FDKP or exenatide/FDKP. These data are surprisingbecause the circulating half-life of exenatide (89 min) is considerablylonger than that of GLP-1 (15 min). Indeed, exenatide was developed tomaximize circulating half-life for the purpose of increasing efficacy.These data suggest that the longer circulating half-life of exenatideoffers no advantage in controlling hyperglycemia when using pulmonaryadministration. Moreover pulmonary administration of either moleculeprovided superior blood glucose control the SC exenatide.

FIG. 7 depicts mean plasma exendin concentrations in male ZDF ratsreceiving exendin-4/FDKP powder formulation administered by pulmonaryinsufflation versus subcutaneous exendin-4. The closed squares representthe response following pulmonary insufflation of exendin-4/FDKP powder.The open squares represent the response following administration ofsubcutaneously administered exendin-4. The data are plotted as ±standarddeviation. The data show that rats insufflated with powders providingGLP-1 doses of 0.12, 0.17, and 0.36 mg produced maximum plasma GLP-1concentrations (C_(max)) of 2.3, 4.9 and 10.2 nM and exposures (AUC) of57.1 nM·min, 92.6 nM·min, and 227.9 nM·min, respectively (t_(max)=10min, t_(1/2)=10 min). In an intraperitoneal glucose tolerance testconducted after 4 consecutive days of dosing 0.3 mg GLP-1 per day,treated animals exhibited significantly lower glucose concentrationsthan the control group (p<0.05). At 30 min post-challenge, glucoseincreased by 47% in control animals but only 17% in treated animals.

FIG. 8 depicts the change in blood glucose from baseline in male ZDFrats receiving either air control, exendin-4/FDKP powder, or GLP-1/FDKPpowder by pulmonary insufflation versus subcutaneous exendin-4 andexendin-4 administered by pulmonary insufflation. The closed diamondsrepresent the response following pulmonary insufflation ofexendin-4/FDKP powder. The closed circles represent the responsefollowing administration of subcutaneous exendin-4. The closed trianglesrepresent the response following administration of GLP-1/FDKP powder.The closed squares represent the response following pulmonaryinsufflation of air alone. The open squares represent the response givenby 2 mg of GLP-1/FDKP given to the rats by insufflation followed by a 2mg exendin-4/FDKP powder administered also by insufflation.

Example 3 Oxyntomodulin/FDKP Powder Preparation

Oxyntomodulin, also known as glucagon-37, is a peptide consisting of 37amino acid residues. The peptide was manufactured and acquired fromAmerican Peptide Company, Inc. of Sunnyvale, Calif. FDKP particles insuspension were mixed with an oxyntomodulin solution, then flash frozenas pellets in liquid nitrogen and lyophilized to produce sample powders.

Six powders were prepared with target peptide content between 5% and30%. Actual peptide content determined by HPLC was between 4.4% and28.5%. The aerodynamic properties of the 10% peptide-containing powderwere analyzed using cascade impaction.

The FDKP solution was mixed with an acetic acid solution containingpolysorbate 80 to form particles. The particles were washed andconcentrated by tangential flow filtration to achieve approximately 11%solids by weight.

FDKP particle suspension (1885 mg×11.14% solids=210 mg FDKP particles)was weighed into a 4 mL clear glass vial. The vial was capped and mixedusing a magnetic stirrer to prevent settling. Oxyntomodulin solution(909 mg of 10% peptide in 2 wt % acetic acid) was added to the vial andallowed to mix. The final composition ratio was approximately 30:70oxyntomodulin:FDKP particles. The oxyntomodulin/FDKP suspension had aninitial pH of 4.00 which was adjusted to pH 4.48 by adding 2-10 μLincrements of 1:4 (v/v) ammonium hydroxide/water. The suspension waspelleted into a small crystallization dish containing liquid nitrogen.The dish was placed in a freeze dryer and lyophilized at 200 mTorr. Theshelf temperature was ramped from −45° C. to 25° C. at 0.2° C./min andthen held at 25° C. for approximately 10 hours. The resultant powder wastransferred to a 4 mL clear glass vial. Total yield of the powder aftertransfer to the vial was 309 mg (103%). Samples were tested foroxyntomodulin content by dissolving the oxyntomodulin preparation insodium bicarbonate and assaying by high pressure liquid chromatographyin a Waters 2695 separations system using deionized with 0.1%trifluoroacetic acid (TFA) and acetonitrile with 0.1% TFA as mobilephases, with the wavelength detection set at 220 and 280 nm. Data wasanalyzed using a Waters Empower™ software program.

Pharmacokinetic and Pharmacodynamic Assessment in Rats.

Male ZDF rats (10/group) were assigned to one of four groups. Animals inthe one group received oxyntomodulin by intravenous injection. Animalsin the other three groups received 5% oxyntomodulin/FDKP powder(containing 0.15 mg oxyntomodulin), 15% oxyntomodulin/FDKP powder(containing 0.45 mg oxyntomodulin), or 30% oxyntomodulin/FDKP powder(containing 0.9 mg oxyntomodulin) by pulmonary insufflation. Bloodsamples were collected from the tail prior to dosing and at varioustimes after dosing for measurement of plasma oxyntomodulinconcentrations (FIG. 9A). Food consumption was also monitored at varioustimes after dosing with oxyntomodulin (FIG. 9B).

FIG. 9A is a graph comparing the plasma concentrations of oxyntomodulinfollowing administration of an inhalable dry powder formulation atvarious amounts in male ZDF rats and control rats receivingoxyntomodulin by intravenous injection. These data show thatoxyntomodulin is absorbed rapidly following insufflation ofoxyntomodulin/FDKP powder. The time to maximum peak circulatingoxyntomodulin concentrations (T_(max)) was less than 15 min in ratsreceiving inhaled oxyntomodulin. This study shows that the half life ofoxyntomodulin is from about 22 to about 25 min after pulmonaryadministration.

FIG. 9B is a bar graph showing cumulative food consumption in male ZDFrats treated with intravenous oxyntomodulin or oxyntomodulin/FDKP powderadministered by pulomonary insufflation compared to control animalsreceiving an air stream. The data show that pulmonary administration ofoxyntomodulin/FDKP reduced food consumption to a greater extent thaneither intravenous oxyntomodulin or air control with a single dose.

In a similar set of experiments, rats received an air stream as control(Group 1) or 30% oxyntomodulin/FDKP powder by pulmonary insufflation.Rats administered oxyntomodulin/FDKP inhalation powder received doses ofeither 0.15 mg oxyntomodulin (as 0.5 mg of oxyntomodulin/FDKP powder;Group 2), 0.45 mg oxyntomodulin (as 1.5 mg of oxyntomodulin/FDKP powder,Group 3) or 0.9 mg oxyntomodulin (as 3 mg of oxyntomodulin/FDKP powder,Group 4) prepared as described above. The studies were conducted in ZDFrats fasted for 24 hr prior to the start of the experiment. Rats wereallowed to eat after receiving the experimental dose. A predeterminedamount of food was given to the rats and the amount of food the ratsconsumed was measured at various times after the start of theexperiment. The oxyntomodulin/FDKP dry powder formulation wasadministered to the rats by pulmonary insufflation and food measurementsand blood samples were taken at various points after dosing.

FIGS. 10A and 10B show circulating oxyntomodulin concentrations for alltest animals and the change in food consumption from control,respectively. Rats given oxyntomodulin consumed significantly less foodthan the control rats for up to 6 hr after dosing. Higher doses ofoxyntomodulin appeared to suppress appetite more significantly that thelower doses indicating that appetite suppression is dose dependent, asthe rats given the higher dose consumed the least amount of food at alltime points measured after dosing.

Maximal concentrations of oxyntomodulin in blood were detected at 10 to30 min and that maximal concentration of oxyntomodulin was dosedependent as the rats receiving 1.5 mg of oxyntomodulin had a maximalplasma concentration of 311 μg/mL and rats receiving 3 mg ofoxyntomodulin had a maximal plasma concentration of 660 μg/mL. Thehalf-life (t_(1/2)) of oxyntomodulin in the Sprague Dawley rats afteradministration by pulmonary insufflation ranged from about 25 to 51 min.

Example 4 Administration of GLP-1 in an Inhalable Dry Powder to Type 2Diabetic Patients

A Phase 1 clinical trial of GLP-1/FDKP inhalation powder was conductedin patients suffering with Type 2 diabetes mellitus to assess theglucose levels of the patients before and after treatment with GLP-1 drypowder formulation by pulmonary inhalation. These studies were conductedaccording to Example 1 and as described herein. GLP-1 inhalation powderwas prepared as described in U.S. patent application Ser. No.11/735,957, which disclosure is incorporated herein by reference. Thedry inhalation powder contained 1.5 mg of human GLP-1(7-36) amide in atotal of 10 mg dry powder formulation containing FDKP in single dosecartridge. For this study, 20 patients with Type 2 diabetes, includingadult males and postmenopausal females, were fasted overnight andremained fasted for a period of 4 hr after GLP-1 inhalation powderadministration. The dry powder formulation was administered using theMedTone® dry powder inhaler (MannKind Corporation), and described inU.S. patent application Ser. No. 10/655,153, which disclosure isincorporated herein by reference in its entirety.

Blood samples for assessing serum glucose levels from the treatedpatients were obtained at 30 min prior to dosing, at dosing (time 0),and at approximately 2, 4, 9, 15, 30, 45, 60, 90, 120 and 240 minfollowing GLP-1 administration. The serum glucose levels were analyzedfor each sample.

FIG. 11 is a graph showing the results of these studies and depicts theglucose values obtained from six fasted patients with Type 2 diabetesfollowing administration of a single dose of an inhalable dry powderformulation containing GLP-1 at various time points. The glucose valuesfor all six patients decreased following administration of GLP-1 andremained depressed for at least 4 hrs after administration at thetermination of the study.

FIG. 12 is a graph showing the mean glucose values for the group of sixfasted patients with Type 2 diabetes whose glucose values are shown inFIG. 11. In FIG. 12, the glucose values are expressed as the mean changeof glucose levels from zero time (dosing) for all six patients. FIG. 12shows a mean glucose drop of approximately 1 mmol/L, which isapproximately equivalent to from about 18 mg/dL to about 20 mg/dL, isattained by the 30 min time point. This mean drop in glucose levels tolast for 120 min. The changes are larger in subjects with higherbaseline glucose and more prolonged, whereas in 2 of the 6 subjects, thesubjects with the lowest baseline fasted blood glucose, showed only atransient lowering of glucose levels in this timeframe (data not shown).It was noted that those with higher fasting glucose do not typicallyhave the same insulin response as those with lower values, so that whenstimulated, those subjects with higher fasting glucose typically exhibita greater response than those whose glucose value are closer to normal.

Example 5 First Pass Distribution Model of Intact GLP-1 to Brain andLiver

First pass distribution of GLP-1 through the systemic circulationfollowing pulmonary delivery and intravenous bolus administration wascalculated to determine the efficacy of delivery for both methods ofGLP-1 administration. A model was developed based on the assumptionsthat: (1) the absorption of GLP-1 from the lungs and into the pulmonaryveins exhibited zero-order kinetics; (2) the distribution of GLP-1 tothe brain and within the brain occurs instantaneously, and (3) clearanceof GLP-1 from the brain and liver distribution is driven by basal bloodflow only. Based on these assumptions, the analysis to determine theamount of GLP-1 in the brain and liver was based on published data withrespect to extraction of GLP-1 by certain tissues and organs (Deacon, C.F. et al. “Glucagon-like peptide 1 undergoes differentialtissue-specific metabolism in the anesthetized pig.” AmericanPhysiological Society, 1996, pages E458-E464), and blood flowdistribution to the body and rate due to cardiac output from humanstudies (Guyton Textbook of Physiology, 10^(th) Edition; W. B. Saunders,2000, page 176). In a normal subject (70 kg) having normal physiologicalparameters such as blood pressure at resting, the basal flow rate to thebrain and liver are 700 mL/min and 1350 mL/min, respectively. Based oncardiac output, blood flow distribution to the body has been calculatedto be 14% to the brain, 27% to the liver and 59% to remaining bodytissues (Guyton).

Using the above-mentioned parameters, the relative amounts of GLP-1 thatwould be distributed to the brain and liver for a 1 mg dose given bypulmonary and intravenous administration were determined. One mg ofGLP-1 was divided by 60 seconds, and the resultant number was multipliedby 14% flow distribution to the brain. Therefore, every second afraction of the dose is appearing in the brain. From the data availableindicating that blood in the brain is equal to 150 mL and the clearancerate is 700 mL/min, the calculations on clearance of GLP-1 yields about12 mL/second, which equals approximately 8% of the blood volume beingcleared from the brain per second. In the intravenous studies in pigsreported by Deacon et al., 40% of GLP-1 was instantaneously metabolizedin the vein and 10% was also metabolized in the deoxygenated blood inthe lung. Accordingly, 40% followed by another 10% of the total GLP-1was subtracted from the total amount administered in the calculationswith respect to the intravenous data analysis.

For the GLP-1 amounts estimated in the liver, the same degradationassumptions were made for the intravenous and pulmonary administrationroutes, with 40% followed by another 10% total amount loss for the IVdose. Twenty-seven percent of the remaining GLP-1 was assumed to bedistributed to the liver, with 75% of the blood passing through theportal bed first. Instantaneous distribution of blood in the liver wasassumed. Calculations were as follows: One mg of GLP-1 was divided by 60seconds, 40% followed by another 10% of the total GLP-1 was subtractedfrom the total amount administered with respect to the intravenous dataanalysis. No degradation was assumed for the pulmonary administration.The resultant numbers were multiplied by 27% flow distribution to theliver, for both routes of administration, with 75% of this amountpassing though the portal bed first. In the intravenous studies in pigsreported by Deacon et al., 20% extraction by the portal bed wasreported; hence 75% of the amount of GLP-1 was reduced by 20% beforebeing introduced into the liver. Therefore, the total amount of GLP-1appearing in the liver every second is comprised of a fraction which hasundergone metabolism in the portal bed. From the data availableindicating that blood volume in the liver is equal to 750 mL and theclearance rate is 1350 mL/minute, the calculations on clearance of GLP-1yields about 22.5 mL/second, which equals approximately 3% of the bloodvolume being cleared from the liver per second. Deacon et al. reported45% degradation in the liver, accordingly, 45% of the total GLP-1 wassubtracted from the total amount appearing in the liver, and theremainder was added to the total remaining amount.

The results of the calculations described above are presented in Tables4 and 5. The calculated GLP-1 distribution in brain and liver afterpulmonary administration (Table 4) are shown below:

TABLE 4 Pulmonary administration of 1 mg GLP-1 Time in Seconds Brain(μg) Liver (μg) 1 2.3 2.10 5 9.94 9.91 60 29.0 58.9

The results illustrating the distribution of GLP-1 after an intravenousbolus administration are shown in Table 5 below:

TABLE 5 Intravenous bolus administration of 1 mg GLP-1 over 1 minuteTime in Seconds Brain (μg) Liver (μg) 1 1.26 1.14 5 5.37 5.35 60 15.631.7

The calculations above are representative illustrations of thedistribution of GLP-1 to specific tissues of the body after degradationof GLP-1 by endogenous enzymes. Based on the above determinations, theamounts of GLP-1 in brain and liver after pulmonary administration areabout 1.82 to about 1.86 times higher than the amounts of GLP-1 afterintravenous bolus administration. Therefore, the data indicate thatpulmonary delivery of GLP-1 can be a more effective route of deliverywhen compared to intravenous administration of GLP-1, as the amount ofGLP-1 at various times after administration would be about double theamount obtained with intravenous administration. Therefore, treatment ofa disease or disorder comprising GLP-1 by pulmonary administration wouldrequire smaller total amounts, or almost half of an intravenous GLP-1dose that is required to yield the same or similar effects.

Example 6

The studies in this example were conducted to measure thepharmacokinetic parameters of various active agents by subcutaneousadministration and in formulations comprising a FDKP, FDKP disodiumsalt, succinyl-substituted-DKP (SDKP, also referred to herein asCompound 1) or asymmetrical (fumaryl-monosubstituted)-DKP (also referredherein as Compound 2) to ZDF rats administered by pulmonaryinsufflation. The rats were divided into 8 groups and five rats wereassigned to each group. Each rat in Group 1 received a 0.3 mg dose ofexendin-4 in phosphate buffered saline solution by pulmonary liquidinstillation; Group 2 received 0.3 mg of exendin-4 in phosphate bufferedsaline by subcutaneous injection.

Rats in Groups 3-8 received their dosing of active agent or exendin-4 bypulmonary insufflation as follows: Group 3 rats received a 2 mgformulation of GLP-1/FDKP by pulmonary insufflation, followed by a 2 mgdose of exendin-4; Group 4 received a formulation of exendin-4/FDKP;Group 5 rats received a 3 mg dose of exendin-4 formulated as a 9.2% loadin a disodium salt of FDKP; Group 6 rats received a 2 mg dose ofexendin-4 formulated as a 13.4% load in a disodium salt of FDKP; Group 7rats received a 2 mg dose of exendin-4 formulated as a 14.5% load inSDKP, and Group 8 rats received a 2 mg dose of exendin-4 formulated as a13.1% load in asymmetrical (fumaryl-mono-substituted) DKP.

The dosing of the animals occurred over the course of two days toaccommodate the high number of subjects. The various test articles wereadministered to the animals and blood samples were taken at varioustimes after dosing. Exendin-4 concentrations were measured in plasmaisolates; the results for which are provided in FIG. 13. As depicted inthe graph, Group 4 treated rats which received exendin-4 in aformulation containing FDKP exhibited high levels of exendin-4 in theblood earlier than 30 min and at higher levels than the rats in Group 2,which received exendin-4 by subcutaneous administration. In all groups,the levels of exendin-4 decrease sharply at about an hour afteradministration.

Administration of exendin-4/FDKP by pulmonary insufflation in ZDF ratshas similar dose-normalized C_(max), AUC, and bioavailability asexendin-4 administered as a subcutaneous injection. Exendin-4/FDKPadministered by pulmonary insufflation showed a greater than two-foldhalf life compared to exendin-4 by subcutaneous injection. Exendin-4administered as a fumaryl(mono-substituted)DKP, or SDKP formulationshowed lower dose normalized C_(max), AUC, and bioavailability comparedto subcutaneous injection (approximately 50% less) but higher levelsthan pulmonary instillation.

After an overnight fast, ZDF rats were given a glucose challenge byintraperitoneal injection (IPGTT). Treatment with exendin-4/FDKP showeda greater reduction in blood glucose levels following the IPGTT comparedto exendin-4 by the subcutaneous route. Compared to air control animals,blood glucose levels were significantly lowered following an IPGTT for30 and 60 min in animals administered exendin-4 by subcutaneousinjection and exendin-4/FDKP powder by pulmonary administration,respectively. Group 3 ZDF rats treated with exendin-4/FDKP and GLP-1 bypulmonary insufflation after intraperitoneal glucose administration(IPGTT) showed surprisingly lower blood glucose levels following IPGTTcompared to either treatment alone at 30 min post dose (−28% versus−24%).

Example 7

The studies in this example were conducted to measure thepharmacokinetic and pharmacodynamic profile of peptide YY(3-36)formulations by pulmonary administration to ZDF rats compared tointravenous injections.

Preparation of PYY/FDKP Formulation for Pulmonary Delivery:

Peptide YY(3-36) (PYY) used in these experiments was obtained fromAmerican Peptide and was adsorbed onto FDKP particles as a function ofpH. A 10% peptide stock solution was prepared by weighing 85.15 mg ofPYY into an 8 ml clear vial and adding 2% aqueous acetic acid to a finalweight of 762 mg. The peptide was gently mixed to obtain a clearsolution. FDKP suspension (4968 mg, containing 424 mg of FDKP preformedparticles) was added to the vial containing the PYY solution, whichformed a PYY/FDKP particle suspension. The sample was placed on amagnetic stir-plate and mixed thoroughly throughout the experiment. Amicro pH electrode was used to monitor the pH of the mixture. Aliquotsof 2-3 μL of a 14-15% aqueous ammonia solution were used toincrementally increase the pH of the sample. Sample volumes (75 μL foranalysis of the supernatant; 10 μL for suspension) were removed at eachpH point. The samples for supernatant analysis were transferred to 1.5ml, 0.22 μm filter tubes and centrifuged. The suspension and filteredsupernatant samples were transferred into HPLC autosampler vialscontaining 990 μL of 50 mM sodium bicarbonate solution. The dilutedsamples were analyzed by HPLC to assess the characteristics of thepreparations. The experiments indicated that, for example, a 10.2% ofPYY solution can be adsorbed onto FDKP particles at pH 4.5 In thisparticular preparation, for example, the PYY content of the resultantpowder was determined by HPLC to be 14.5% (w/w). Cascade measurements ofaerodynamic characteristics of the powder showed a respirable fractionof 52% with a 98% cartridge emptying when discharged through theMedTone® dry powder inhaler (MannKind Corporation). Based on the resultsabove, multiple sample preparations of PYY/FDKP powder were made,including, 5%, 10%, 15% and 20% PYY.

Pharmacokinetic and Pharmacodynamic Studies:

Female ZDF rats were used in these experiments and divided into 7groups; five rats were assigned to each group, except for Group 1 whichhad 3 rats. The rats were fasted for 24 hr prior to being given theirassigned dose and immediately provided with food after dosing andallowed to eat as desired for the period of the experiment. Each rat inGroup 1 received a 0.6 mg IV dose of PYY in phosphate buffered salinesolution; Group 2 rats received 1.0 mg of PYY pulmonary liquidinstillation; Group 3 rats were designated as control and received astream of air; Groups 4-7 rats received a dry powder formulation forinhalation administered by pulmonary insufflation as follows: Group 4rats received 0.15 mg of PYY in a 3 mg PYY/FDKP powder formulation of 5%PYY (w/w) load; Group 5 rats received 0.3 mg of PYY in a 3 mg PYY/FDKPpowder formulation of 10% PYY (w/w) load; Group 6 rats received 0.45 mgof PYY in a 3 mg PYY/FDKP powder formulation of 15% PYY (w/w) load;Group 7 rats received 0.6 mg of PYY in a 3 mg PYY/FDKP powderformulation of 20% PYY (w/w) load.

Food consumption was measured for each rat at 30, 60, 90, 120, 240 minand 24 hr after dosing. PYY plasma concentrations and glucoseconcentrations were determined for each rat from blood samples takenfrom the rats before dosing and at 5, 10, 20, 30, 45, 60 and 90 minafter dosing. The results of these experiments are shown in FIGS. 14-16and Table 6 below. FIG. 14 is a bar graph of representative data fromexperiments measuring food consumption in female ZDF rats receiving PYYformulations by intravenous administration and by pulmonaryadministration in a formulation comprising a fumaryl-diketopiperazine atthe various doses. The data show that food consumption was reduced forall PYY-treated rats when compared to control with the exception ofGroup 2 which received PYY by instillation. Reduction in foodconsumption by the rats was statistically significant for the ratstreated by pulmonary insufflation at 30, 60, 90 and 120 min afterPYY-dosing when compared to control. The data in FIG. 14 also show thatwhile IV administration (Group 1) is relatively effective in reducingfood consumption in the rats, the same amount of PYY (0.6 mg)administered by the pulmonary route in an FDKP formulation (Group 7) wasmore effective in reducing the amount of food intake or suppressingappetite for a longer period of time. All PYY-treated rats receivingpulmonary PYY-FDKP powders consumed less food when compared to controls.

FIG. 15 depicts the measured blood glucose levels in the female ZDF ratsgiven PYY formulations by IV administration; by pulmonary administrationwith various formulations comprising a fumaryl-diketopiperazine and aircontrol rats. The data indicate the blood glucose levels of thePYY-treated rats by pulmonary insufflation remained relatively similarto the controls, except for the Group 1 rats which were treated with PYYIV. The Group 1 rats showed an initial lower blood glucose level whencompared to the other rats up to about 15 min after dosing.

FIG. 16 depicts representative data from experiments measuring theplasma concentration of PYY in the female ZDF rats given PYYformulations by IV administration; by pulmonary administration withvarious formulations comprising a fumaryl-diketopiperazine, and aircontrol rats taken at various times after administration. Thesemeasurements are also represented in Table 6. The data show that Group 1rats which were administered PYY IV attained a higher plasma PYYconcentration (30.7 μg/mL) than rats treated by pulmonary insufflation.Peak plasma concentration (T_(max)) for PYY was about 5 min for Groups1, 6 and 7 rats and 10 min for Group 2, 4 and 5 rats. The data show thatall rats treated by pulmonary insufflation with a PYY/FDKP formulationhad measurable amounts of PYY in their plasma samples, however, theGroup 7 rats had the highest plasma PYY concentration (4.9 μg/mL) andvalues remained higher than the other groups up to about 35 min afterdosing. The data also indicate that the plasma concentration of PYYadministered by pulmonary insufflation is dose dependent. Whileadministration by IV injection led to higher venous plasma concentrationof PYY than did pulmonary administration of PYY/FDKP at the dosagesused, the greater suppression of food consumption was nonethelessachieved with pulmonary administration of PYY/FDKP.

TABLE 6 Rat Group T½ Tmax Cmax AUCall/D Number (min) (min) (μg/mL)(min/mL) BA (%) 1 13 5 30.7 0.61 100% 2 22 10 1.7 0.06 11 4 23 10 0.510.10 16 5 30 10 1.33 0.15 25 6 26 5 2.79 0.20 33 7 22 5 4.90 0.22 36

FIG. 17 illustrates the effectiveness of the present drug deliverysystem as measured for several active agents, including insulin,exendin, oxyntomodulin and PYY and exemplified herewith. Specifically,FIG. 17 demonstrates the relationship between drug exposure andbioeffect of the pulmonary drug delivery system compared to IV and SCadministration of the aforementioned active agents. The data in FIG. 17indicate that the present pulmonary drug delivery system provides agreater bioeffect with lesser amounts of drug exposure than intravenousor subcutaneous administration. Therefore, lesser amounts of drugexposure can be required to obtain a similar or greater effect of adesired drug when compared to standard therapies. Thus, in oneembodiment, a method of delivering an active agent including, peptidessuch as GLP-1, oxyntomodulin, PYY, for the treatment of disease,including diabetes, hyperglycemia and obesity which comprisesadministering to a subject in need of treatment an inhalable formulationcomprising one or more active agents and a diketopiperazine whereby atherapeutic effect is seen with lower exposure to the active agent thanrequired to achieve a similar effect with other modes of administration.In one embodiment, the active agents include peptides, proteins,lipokines.

Example 8 Assessment of GLP-1 Activity in Postprandial Type 2 DiabetesMellitus

The purpose of this study was to evaluate the effect of a GLP-1 drypowder formulation on postprandial glucose concentration and assess itssafety including adverse events, GPL-1 activity, insulin response, andgastric emptying.

Experimental Design: The study was divided into two periods and enrolled20 patients diagnosed with type 2 diabetes ranging in age from 20 to 64years of age. Period 1 was an open-label, single-dose, trial in which 15of the patients received a dry powder formulation comprising 1.5 mg ofGLP-1 in FDKP administered after having fasted overnight. As control, 5subjects received FDKP inhalation powder after fasting overnight. Period2 was performed after completion of Period 1. In this part of the study,the patients were given 4 sequential treatments each with a mealchallenge consisting of 475 Kcal and labeled with ¹³C-octanoate asmarker. The study was designed as a double-blind, double dummy,cross-over, meal challenge study, in which saline as control andexenatide were given as injection 15 minutes before a meal and drypowder formulations of inhalable GLP-1 or placebo consisting of a drypowder formulation without GLP-1, were administered immediately beforethe meal and repeated 30 minutes after the meal. The four treatmentswere as follows: Treatment 1 consisted of all patients receiving aplacebo of 1.5 mg of dry powder formulation without GLP-1. In Treatment2, all patients received one dose of 1.5 mg of GLP-1 in a dry powderformulation comprising FDKP. In Treatment 3, all patients received twodoses of 1.5 mg of GLP-1 in a dry powder formulation comprising FDKP,one dose immediately before the meal and one dose 30 minutes after themeal. In Treatment 4, the patients received 10 μg of exenatide bysubcutaneous injection. Blood samples from each patient were taken atvarious times before and after dosing and analyzed for severalparameters, including GLP-1 concentration, insulin response, glucoseconcentration and gastric emptying. The results of this study aredepicted in FIGS. 18-20.

FIG. 18 depicts the mean GLP-1 levels in blood by treatment group asdescribed above. The data demonstrate that the patients receiving thedry powder formulation comprising 1.5 mg of GLP-1 in FDKP hadsignificantly higher levels of GLP-1 in blood soon after administrationas shown in panels A, B and C and that the levels of GLP-1 sharplydeclined after administration in fed or fasted individuals. There wereno measurable levels of GLP-1 in the exenatide-treated group (Panel D),or in controls (Panel E) receiving the dry powder formulation.

FIG. 19 depicts the insulin levels of the patients in the study beforeor after treatment. The data show that endogenous insulin was producedin all patients after treatment including the placebo-treated patientsin the meal challenge studies (Panel B), except for the fasted controlpatients (Panel C) who received the placebo. However, the insulinresponse was more significant in patients receiving GLP-1 in a drypowder composition comprising FDKP, in which the insulin response wasobserved immediately after treatment in both fed and fasted groups(Panels D-F). The results also showed that the glucose levels werereduced in patients treated with the dry powder formulation of GLP-1.Administration of the dry powder formulation of GLP-1 resulted in adelayed rise in blood glucose and reduced overall exposure (AUC) toglucose. Both the delayed rise and lessened exposure were morepronounced in subjects receiving a second administration of GLP-1inhalation powder (data not shown). The magnitude of insulin releasevaried among patients, with some showing small but physiologicallyrelevant levels of insulin whereas others exhibited much larger insulinreleases. Despite the difference in insulin response between thepatients, the glucose response was similar. This difference in insulinresponse may reflect variations in degree of insulin resistance anddisease progression. Assessment of this response can be used as adiagnostic indicator of disease progression with larger releases(lacking greater effectiveness at controlling blood glucose levels)indicating greater insulin resistance and disease progression.

FIG. 20 depicts the percent gastric emptying by treatment groups. PanelA (patients in Treatment 3) and Panel B (patients in Treatment 2)patients had similar gastric emptying characteristics or percentages asthe control patients shown in Panel D (Placebo treated patients with adry powder formulation comprising FDKP without GLP-1). The data alsoshow that patients treated with exenatide even at a 10 μg dose exhibiteda significant delay or inhibition in gastric emptying when compared tocontrols. The data also demonstrate that the present system fordelivering active agents comprising FDKP and GLP-1 lacks inhibition ofgastric emptying; induces a rapid insulin release following GLP-1delivery and causes a reduction in glucose AUC levels.

Example 9 Preparation and Characterization of Sumatriptan-DisodiumFumaryl Diketopiperazine (Sumatriptan-Na₂FDKP) Inhalation Dry Powder

Sumatriptan-Na₂FDKP powder was prepared from commercially availabletablets of sumatriptan succinate. Sumatriptan succinate (Imitrex®,GlaxoSmithKline) was extracted from crushed tablets suspended in HPLCgrade water to form a solution. The solution was then filtered through0.45 μm nylon syringe filters to remove undissolved excipients and thefiltrate was processed through a quaternary amine ion exchangeextraction column to eliminate the succinate group. The sumatriptan insolution at a concentration greater than 10 mg/mL was then combined witha solution of Na₂FDKP at a concentration greater than about 10 mg/mL insolution. The Na₂FDKP was either prepared earlier or prepared from theFDKP free acid by dissolution with two equivalents of sodium hydroxide.In some experiments, the ratio of the concentration of sumatriptanstarting solution to FDKP was greater than 1. The solution was spraydried (Büchi Mini Spray Dryer Model B-290) at an inlet temperature offrom about 145° C. to about 200° C. and an outlet temperature of fromabout 75° C. to about 85° C. The drying gas was nitrogen set at a flowrate of about 670 L/hr. A dried powder was obtained.

To ascertain the amount of sumatriptan in the powder, a sample of thepowder was dissolved in an HPLC solution and assayed for content using ahigh pressure liquid chromatography (HPLC) system. The HPLC methodquantifies sumatriptan in the presence of FDKP. FIG. 21 depicts an HPLCchromatogram of a solution of the dissolved powder, which shows that thetwo compounds can be easily separated and identified. Powders were alsotested by thermal analysis (TGA and DSC) and cascade impaction. Table 7shows representative data of Sumatriptan-Na₂FDKP powder characteristicsmade by the instant method, wherein the ratio of sumatriptan succinateconcentration to FDKP in solution was about 1.5.

TABLE 7 Assay (wt %) Sumatriptan % Cascade impaction (% by weight) FDKPLOD Total % RF/fill % CE 38.8* 37.0 3.4 79.2 17.9 68.0 *TargetSumatriptan level = 40%

Sumatriptan-Na₂FDKP powders made by the present method contained up toabout 40% by weight of sumatriptan. The water content determined by losson drying (LOD) was about 3.4%. The percent respirable fraction per fillwas about 18%, and the amount of powder delivered by or emitted from theinhaler (cartridge emptying, CE) was about 68% using a using a breathpowered inhaler as described in U.S. patent application Ser. No.12/473,125 (US 2009/0308390), which disclosure is incorporated byreference in its entirety.

Table 8 illustrates the characterization of the bulk dry powder.

TABLE 8 Assay (wt %) Sumatriptan % Cascade impaction (% by weight) FDKPLOD Total % RF/fill % CE 38.8* 37.0 3.4 79.2 17.9 68.0 *TargetSumatriptan level = 40%

Example 10 Sumatriptan-Na₂FDKP Inhalation Dry Powder Pharmacokinetic(PK) and Pharmacodynamic (PD) Studies in Rats

Powder Preparation and Characterization:

Sumatriptan-Na₂FDKP powder was prepared as described in Example 9 above,except that the sumatriptan succinate was purchase from LGM Pharma (BocaRaton, Fla.) and L-leucine was added to study whether the aerodynamicperformance of the resulting spray-dried powder formed would beimproved. Three feed solutions were prepared at 4.5% total solidsconcentration for a 5 g scale. The feed solutions were prepared byadding FDKP disodium salt, sumatriptan succinate, and L-leucine (0-20 wt%) to de-ionized water with mixing. The solutions were titrated withdilute aqueous ammonia to pH 6.00. The resulting clear feed solutionswere vacuum filtered through a 0.2 μm PES filter membrane and spraydried as described in Example 9, however, the drying gas flow was set at25 kg/hr, the atomization flow was about 4 kg/hr and the atomizationpressure was set at 4 bar. The sumatriptan succinate concentration (drybasis) in each solution was 56% to obtain a 40% sumatriptan target load.The powders were analyzed by HPLC, cascade impaction, Karl Fischertitration, scanning electron microscopy (SEM) and tap and bulk density.The results of these studies are shown in Table 9 and FIG. 22.

TABLE 9 Bulk Tapped % L- Sumatriptan % RF/ % % density density leucineAssay (wt %) fill CE water g/mL g/mL 0 39.8 9.8 58.8 1.9 0.25 0.38 1037.6 61.3 93.3 5.2 0.18 0.38 20 38.8 63.3 88.2 4.4 0.18 0.31

The data in Table 9 illustrate that the target and measured sumatriptancontent for the bulk sumatriptan-Na₂FDKP powders are comparable.Aerodynamic performance improved with the addition of leucine. Thepowder without leucine had an RF/fill of 9.8% with 58.8% CE, theaddition of 10% leucine increased RF/fill to 61.3% with 93.3% CE, andthe addition of 20% leucine increased RF/fill to 63.3.% with 88.2% CE.The leucine-containing sumatriptan-Na₂FDKP powders had higher residualwater content than the leucine-free powder. The addition of leucine alsoreduced the bulk powder density by approximately 30%.

FIG. 22 is a scanning electron micrograph of the three powderscharacterized in Table 3. As shown in panels A-F, each powder haddistinct morphology. The particles without leucine were fused fragmentswith no uniform shape (Panel A and B). The particles with 10% leucine(Panel C and D) were substantially spherical with smooth surfaces andthe particles made with 20% leucine (Panel E and F) have a raisin-likeor shriveled morphology, typical of insulin-FDKP salt powders.

Stability of Sumatriptan-Na₂FDKP Powders:

Powders were also tested to determine their degree of stability. Samplesof powder were incubated for a period of three months in an open dishexposed at 25° C./60% relative humidity (RH) and at 40° C./70% RH.Samples of the powders were assay by the HPLC method at 1, 2 and 3months after the start of the experiments. The results are presented inTables 10 and 11 below.

TABLE 10 Storage Condition: 25° C./60% RH Sumatriptan Assay Initial 1month 2 months 3 months 0% L-leucine powder 39.8 38.9% 39.9% 39.6%(100%)  (98%) (100%)  (98%) 10% L-leucine powder 37.6 39.1% 40.0% 39.0%(100%) (104%) (106%) (104%) 20% L-leucine powder 38.8 38.9% 40.2% 39.4%(100%) (100%) (104%) (102%)

TABLE 11 Storage Condition: 40° C./75% RH Sumatriptan Assay Initial 7days 14 days 0% L-leucine powder 39.8 38.0 37.8 (100%)  (95%)  (95%) 10%L-leucine powder 37.6 38.8 38.6 (100%) (103%) (103%) 20% L-leucinepowder 38.8 39.3 39.3 (100%) (101%) (101%)

The data show that there was no degradation of the sumatriptan in thecomposition even after three months of exposure to 25° C./60% RH with orwithout L-leucine. At higher temperature, 40° C./70% RH, however, aninsignificant, but slight decrease in sumatriptan content is observedafter 1 and 2 weeks of incubation when compared to the samplescontaining L-leucine.

Inhalation Studies in Rats Using Sumatriptan-Na₂FDKP Powders:

Powders prepared as described above were used in these experiments. ThePK profile of sumatriptan administered as sumatriptan-Na₂FDKP powder(37.4% sumatriptan by weight) by pulmonary insufflation was evaluatedand compared to sumatriptan nasal spray administered by pulmonaryinstillation or sumatriptan administered by intravenous injection orsubcutaneous injection in female Sprague Dawley rats (n=6/group) (Table12).

TABLE 12 Achieved Sumatriptan Dose Group Test Article (mg) 1 Air Control0 2 Sumatriptan by intravenous injection 0.358 3 Sumatriptan bysubcutaneous injection 0.358 4 Sumatriptan nasal spray by pulmonary0.483 instillation 5 Sumatriptan-Na₂FDKP powder by 0.169* pulmonaryinsufflation *mean achieved dose

Blood samples for sumatriptan analysis were collected before dosing andat 2, 5, 10, 15, 30, 60, 90, 120 and 240 minutes after dosing. Animalswere divided into two subsets (n=3/timepoint) for blood collection.Sumatriptan in serum was analyzed using an established LCMS assay.Maximum concentration and bioavailability of sumatriptan insufflated assumatriptan-Na₂FDKP powder was higher than the sumatriptan administeredby liquid instillation (nasal spray formulation) and comparable tosumatriptan administered by subcutaneous injection (FIG. 23). FIG. 23shows that the time to maximum concentration was 5 minutes in theinsufflation group versus 15 minutes in the pulmonary liquidinstillation group. Overall dose-normalized exposure was similar forsumatriptan pulmonary insufflation and pulmonary liquid instillation,but the PK profiles are quite different. Sumatriptan was well toleratedacross all treatment groups. Pharmacokinetic parameters were calculatedusing noncompartmental methods and the nonlinear regression programWinNonlin v5.2 (Table 12) based on the mean concentration curve(n=3/time point/formulation) after correction for the actualadministered dose. Table 13 summarizes representative pharmacokineticdata in female Sprague Dawley rats.

TABLE 13 AUC C_(max) (min, ng/ ng/ t_(max) t_(1/2) Bioavailability GroupRoute mL)/mg mL/mg min min % 2 IV 273,729 26,719 2 30.4 100 3 SC 99,5832309 5 36.6 36.4 4 LIS 70,063 776 15 45.8 25.6 5 INS 69,411 2145 5 2425.4 *IV = Intravenous injection; SC = subcutaneous injection; LIS =Pulmonary liquid instillation; INS = pulmary insufflation

It is apparent that the bioavailability of sumatriptan administered asNa₂FDKP sumatriptan powder by pulmonary insufflation was comparable tosumatriptan nasal spray administered by liquid instillation, but its PKprofile (t_(max), C_(max)) resembled SC injection.

Example 11 Sumatriptan-Na₂FDKP Inhalation Dry Powder PK and PD Studiesin Beagle Dogs

Pharmacodynamic Study: The pharmacodynamic and pharmacokinetic profilesof sumatriptan were evaluated in an accepted migraine model inanesthetized dogs. The pathogenesis of migraine is primarily due to amarked and prolonged period of vasodilation of cranial vessels. A modelof migraine was induced by a single intra-arterial injection ofcapsaicin which produces carotid vasodilation. Animals received eitherair control (n=2), sumatriptan by intranasal instillation using amicrosprayer (0.28 mg/kg; n=3), sumatriptan-Na₂FDKP powder by pulmonaryinsufflation (0.28 mg/kg sumatriptan; n=3) or sumatriptan by intravenousbolus injection into a peripheral vessel (0.03 mg/kg; n=2). Heart rate,systolic, diastolic, mean arterial blood pressures, and carotid bloodflow and diameter (mean, maximum, minimum flow) were monitored andrecorded continuously. Data were collected continuously and reported as1-minute averages at specific time points after sumatriptanadministration. The study summary is presented in Tables 15 and theresults are shown in FIG. 25.

TABLE 15 Experimental design Dose Group No. of Model Levels Dose No.Animals Induction Test Material (mg/kg) Regimen^(a,b) Monitoring Period1 2 Capsaicin Control 0 Intra-tracheal 30 minutes of stable administered(insufflator) baseline followed by 5 2 3 intra-arteriallySumatriptan^(c) 0.28 Nasal spray minutes of monitoring via a 1-minute(microsprayer) after administration of 3 3 infusion Sumatriptan 0.29dIntra-tracheal capsaicin and prior to (56 μg/min; Na₂FDKP (insufflator)sumatriptan 1 mL/min) powder administration. 4 2 Sumatriptan^(c) 0.030Intravenous Monitoring continued through at least 3 hours after dosingwith test article ^(a)Animals were anesthetized during all dosingprocedures. ^(b)Dosing with the appropriate test article commenced 5minutes after model induction. ^(c)Commercially available product(Imitre ®). dEquivalent to 0.75 mg/kg powder dose based on 38% contentof active ingredient Sumatriptan-Na₂FDKP powder.

Blood samples from the dogs for sumatriptan analysis were collectedbefore dosing and at 2, 5, 10, 15, 30, 60, 90, 120 and 240 minutes afterdosing. Sumatriptan in serum was analyzed using an established LCMSassay.

Based on the PK data, one animal in the sumatriptan-Na₂FDKP powdergroup-treated appeared not to have not received test article, presumablydue to technical difficulties. This animal showed unusually markedvasoconstriction which was suspect. Another animal in this group showedpoor vasoconstriction and high levels of sumatriptan exposure. It wasassumed that the tubes for blood samples from these two animals wereinadvertently switched during collection. Therefore, data presentedherewith were evaluated with (n=3) and without (n=2) the mis-dosedanimal. Both sets of data suggest similar results.

Blood pressure and heart rate were unaltered by the administration ofsumatriptan or control article, regardless of the route ofadministration. Systemic exposure of sumatriptan was associated withreductions in vasodilation. All groups, including the control, hadreduction in carotid artery diameters from the end of capsaicinadministration through 3 hours after dosing. Insufflation ofsumatriptan-Na₂FDKP powder resulted in a more pronounced constriction ofthe carotid artery than the intranasal and intravenous routes ofadministration. The magnitude of vasoconstriction varied significantlybetween dose groups, so the data were analyzed in terms of vesseldiameter relative to baseline diameter, or as a change in vesseldiameter from the end of capsaicin dosing, or from baseline.

The pharmacokinetic profiles of sumatriptan administered assumatriptan-Na₂FDKP powder, nasal spray, or intravenous injection (FIG.24) were consistent with the previous PK study in rats. FIG. 24 depictsthe pharmacokinetic profile of sumatriptan FDKP salt powder (38%sumatriptan) administered by pulmonary insufflation, sumatriptanadministered by nasal instillation, and intravenous injection in femaledogs wherein the data are plotted as ±SD. The data show that the time tomaximum mean peak circulating sumatriptan concentrations (T_(max)) was 5minutes for the sumatriptan-FDKP salt powder and 60 minutes for nasalinstillation. Even though C_(max) and bioavailability were much lowerfor the sumatriptan-Na₂FDKP powder, animals insufflated withsumatriptan-Na₂FDKP exhibited a similar but faster pharmacodynamicresponse than those receiving the nasal spray.

FIG. 25 shows results from these experiments. The data indicate thatreduction in vessel diameter from the end of capsaicin to the end ofexperiment was largest in the group treated with the sumatriptan-Na₂FDKPpowder. The variability in initial vasodilation between groupscomplicates the analysis, but Group 2 (nasal spray) and group 3(sumatriptan-Na₂FDKP powder) responded comparably to capsaicin. Thegroup treated with sumatriptan-Na₂FDKP powder experienced a larger netconstriction in blood vessels and, moreover, the effect had a fasteronset of action.

While the invention has been particularly shown and described withreference to particular embodiments, it will be appreciated thatvariations of the above-disclosed and other features and functions, oralternatives thereof, may be desirably combined into many otherdifferent systems or applications. Also that various presentlyunforeseen or unanticipated alternatives, modifications, variations orimprovements therein may be subsequently made by those skilled in theart which are also intended to be encompassed by the following claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

We claim:
 1. A method of treating symptoms associated with migrainecomprising: administering to a subject in need of treatment for migrainesymptoms a dry powder pharmaceutical composition comprising an effectiveamount of a serotonin receptor agonist and a diketopiperazine or a saltthereof.
 2. The method of claim 1, wherein the dry powder pharmaceuticalcomposition is administered by inhalation.
 3. The method of claim 1,wherein said diketopiperazine is2,5-diketo-3,6-di(4-X-aminobutyl)piperazine; wherein X is selected fromthe group consisting of succinyl, glutaryl, maleyl, and fumaryl; or apharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein the dry powder pharmaceutical composition is manufactured as aunit dose for oral inhalation.
 5. The method of claim 4, wherein saidinhalable dry powder pharmaceutical composition further comprises apharmaceutically acceptable carrier or excipient.
 6. The method of claim4, wherein the dry powder formulation is administered to said subject bypulmonary inhalation using a breath powered, dry powder inhalationsystem.
 7. The method of claim 1, wherein the serotonin receptor agonistis for treating moderate to severe head pain associated with themigraine.
 8. The method of claim 1, wherein the serotonin receptoragonist is a triptan.
 9. The method of claim 8, wherein the triptan isselected from the group consisting of sumatriptan, almotriptan,eletriptan, frovatriptan, naratriptan, rizatriptan, zolmitriptan andpharmaceutically acceptable salts thereof,
 10. The method of claim 8,wherein the triptan is sumatriptan succinate.
 11. The method of claim 8,wherein the triptan is rizatriptan benzoate.
 12. The method of claim 1,wherein the dry powder pharmaceutical composition further comprises analiphatic amino acid.
 13. The method of claim 12, wherein the aliphaticamino acid is selected from the group consisting of alanine, glycine,leucine, isoleucine, norleucine, and serine.
 14. The method of claim 12,wherein the aliphatic amino acid is about 0.5% to about 30% by weight ofthe composition.
 15. The method of claim 1, wherein the dry powderpharmaceutical composition further comprises L-leucine.
 16. The methodof claim 1, wherein the dry powder pharmaceutical composition furthercomprises an active agent.
 17. The method of claim 1, wherein thediketopiperazine isbis-3,6[(N-fumaryl-4-aminobutyl]-2,5-diketopiperazine or a salt thereof.18. The method of claim 17, wherein the diketopiperazine isbis-3,6-[(N-fumaryl-4-aminobutyl)]-2,5-diketopiperazine disodium salt.19. The method of claim 6, wherein the breath powered, dry powderinhalation system comprises a cartridge containing said dry powderpharmaceutical composition.
 20. The method of claim 16, wherein theactive agent is a vasoconstrictor.